CN104853773A

CN104853773A - Methods and compositions for treating and diagnosing acute myocardial infarction

Info

Publication number: CN104853773A
Application number: CN201380033484.3A
Authority: CN
Inventors: 斯罗伯丹·维基斯维奇; 洛沃卡·格古列维奇; 伊沃·杜米克－库勒
Original assignee: Gene Studies Co
Current assignee: Gene Studies Co
Priority date: 2012-04-25
Filing date: 2013-04-25
Publication date: 2015-08-19
Also published as: HK1201732A1; JP6133402B2; HK1213495A1; CA2870365A1; EP2841100A1; US20150104455A1; WO2013163479A1; JP2015520736A; AU2013251442A1; EP2841100A4; AU2013251442B2; NZ631639A

Abstract

Compositions and methods for treating and diagnosing acute myocardial infarction are described. The invention also provides a method of treating an individual to prevent or inhibit damage to myocardial tissue from an acute myocardial infarction comprising administering to the individual an antibody to BMP-1-3, or an antibody to BMP-1-4, or a combination of an antibody BMP-1-3 and an antibody to BMP-1-4 prior to AMI.

Description

Be used for the treatment of the method and composition with diagnosing acute myocardial infarction

with the cross reference of related application

The priority of the U.S. Provisional Application that this application claims the U.S. Provisional Application numbers 61/638,373 submitted on April 25th, 2012 and submit on April 25th, 2012 numbers 61/638,424, the content whole of described U.S. Provisional Application is incorporated herein.

Invention field

The field of the invention is heart disease.Particularly, the invention provides the method and composition be used for the treatment of with diagnosing acute myocardial infarction, it comprises one or more antibody for one or more BMP-1 isotypes.

background of invention

Acute myocardial infarction (" AMI ") is the main cause of death of developed country, and accounts for the death in the whole world 13%.AMI is also referred to as " heart attack ", is when coronary artery or blood vessel become a class heart disease of blocking and causing occurring when losing the blood supply of cardiac muscular tissue.No longer receive cardiac muscular tissue's quick death of enough blood (hypoperfusion), and replaced by weak function or non-functional fibrous scar tissue, described fibrous scar tissue can be expanded, and cause the function cardiac muscular tissue increased to lose, this can cause again cardiac function to be lacked of proper care successively.Have every year in the U.S. and experience first time AMI more than 500,000 people, and the people suffering from myocardial infarction more than 200,000 cures pre hospital time in arrival.

Complicated relation in the cell component of heart and non-cell components, drives the recovery after suitable heart development, stable state and pathology damage (such as AMI).Myocardial cell, fibroblast and endotheliocyte differential expression and respond specific extracellular matrix factors, described extracellular matrix factors promotes intercellular communication and overall cardiac function.Extracellular matrix (" ECM ") promotes machinery, electricity and chemical signal during stable state and growth course.These Signal Regulation cellular activities, such as cell proliferation, migration, adhesion and changes in gene expression.During multiple physiology's heart state, there is different cells and ECM expression change.See people such as Bowers, j. Molec. Cell. Cardiol., 48:474-482(2010).Such as, during myocardial infarction, myocardial cell experience apoptosis, fibroblast experiences intensive propagation, vessel density reduces, and type i collagen, III Collagen Type VI, IV Collagen Type VI, fibronectin and POSTN expression increase causes fibrosis to strengthen and decreased cardiac function.These processes have detrimental effect to left ventricular function, therefore form the treatment basis using antifibrotic agents to suppress or reverse this adverse effect.See people such as such as Sun, cardiovasc. Res., 46:250-256(2000); Jugdutt, circulation, 108:1395-1403(2003); The people such as Lopez, am. J. Physiol. Heart. Circ. Physiol., 299:H1-H9(2010) etc.

If application fast, then normally effective to the treatment of AMI after coronary vasodilator blocks.Positive thrombolytic therapy comprises the medicine of thrombus (clot) or primary angioplasty and support.After chronic, heart infarction, treatment comprises Angiotensin-Converting (" ACE ") inhibitor, beta-blocker, diuretic and calcium-channel antagonists, it can reduce aortic pressure, thus reduce the remodeling ventricle of left ventricle (LV), described remodeling ventricle otherwise can expand infarct size, causes more nonfunctional scar tissue.Open heart operations method comprises repairs or replaces the coronary artery bypass surgery of coronary vasodilator blocked, and make that the nonfunctional infarcted region of heart tissue is repaired, atrophy or the method that removes.

Bone morphogenetic protein-1 (" BMP-1 ", " BMP1 ") initial separation from highly purified BMP Os Bovis seu Bubali extract, and be reported at first subcutaneous (dystopy) bone formation measure in vivo inducing cartilage formed ((people such as Wozney, science, 242: 1528-1534(1988)).But, BMP-1(SEQ ID NO:1) do not share significant amino acid sequence homology with other BMP, BMP-1 does not demonstrate the signal peptide characteristic, predomain, carboxyl terminal (mature structure territory) or the cysteine knot that find in other BMP yet, and other BMP described are TGF beta superfamily members of somatomedin.The error condition of the BMP-1 in TGF-'beta ' family result from osteogenetic primitive organism measure in the flaw (people such as Wozney, (1988)), the cartilage wherein observed in bioassay look like pollute insoluble bone matrix before growth plate cartilage, it is accredited as the tissue that recently formed (see Reddi by mistake, Science, 271:463(1996)).As shown later, BMP-1 can not inducing cartilage or bone formation in the heterotopic osteogenesis of standard measures.See such as, International Patent Publication No. WO 2008/011193 A2.

In fact, BMP-1 display is identical with precollagen C protein enzyme, and described precollagen C protein enzyme is zinc metalloprotein enzyme, cuts the carboxyl predomain of precollagen I, II and III, with produce major fibrous collagen I, II and III ripe monomer (people such as Kessler, science, 271: 360-362(1996); The people such as Li, proc. Natl. Acad. Sci. USA, 93:5127-5130(1996)); For the insoluble collagen in extracellular matrix (ECM) proper mating and as find the fibrous scar tissue relevant to multiple organ disease formed required step (people such as Turtle, expert Opin. Ther. Patents, 14(8): 1185-1197(2004)).Except the effect in its before cutting collagen, BMP-1 also cuts other ECM macromole, comprise front lysyloxidase (prolysyl oxidase) (people such as Panchenko, j. Biol. Chem., 271:7113-7119(1996)), front Biglycan (probiglycan) (people such as Scott, j. Biol. Chem., 275:30504-30511(2000)) and the people such as front laminin，LN-5(Amano, j. Biol. Chem., 275:22728-22735(2000)).BMP-1 also makes IGF1 and its associated proteins and other somatomedin dive from it to discharge in the composite (Muir and Greenspan, j. Biol. Chem., 286(49): 41905-41911(2011)).BMP-1 protein domain structure comprises N-terminal predomain, subsequently for relate to numerous protein protein interaction conservative protein enzyme domains (people such as Bork, j. Mol. Biol., 231:539-545(1993)).The C-terminal of protease domain is CUB and EGF domain.The BMP-1 CUB domain (" CUB1 ") of most N-terminal can cut chordin (chordin), described chordin be protection BMP-2 and BMP-4 avoid activating bmp antagonist (people such as Petropoulou, j. Biol. Chem., 280:22616-22623(2005)), and EGF domain is in conjunction with calcium ion (Ca ²⁺), and can to a part of imparting mechanism rigidity of BMP-1 isotype (people such as Werner j. Mol. Biol., 296:1065-1078(2000)).

BMP1 gene and fruit bat ( drosophila) gene tolloid(" TLD ") relevant, due to the ability of its activation TGF-β sample morphogen (morphogen), described tolloid involves by decapentaplegic(" DPP ") survival rate of Gene Handling.BMP-1 albumen is known as form required during survival rate cascade at present and occurs one of control point (Ge and Greenspan, Birth Defect Res., 78:47-68(2006)).The mice that BMP1 gene is invalid is that perinatal stage is lethal, the failure of closing with veutro body wall and persistence enterocele are formed, may be limited damage people such as (, Development, 122:3587-3595(1996) Suzuki due to the ECM of defect and back of the body abdomen survival rate).Consistent with pCP loss of activity, BMP1 null mice has abnormal collagen fiber.

BMP-1 is the prototype of the little subgroup of the metalloproteases found in the species of broad range.In mammal, there is relevant (or " BMP-1/TLD sample ") metalloproteases of four kinds of BMP-1/TLD.The gene of encoding BMP-1 is also encoded the longer protease of the second, and it is encoded by alternatively spliced mRNA.Have the domain constructs substantially the same with TLD, this protease is appointed as mammal Toll oid(" mTLD ") (people such as Takahara, j. Biol. Chem., 269:32572-32578(1994)).In addition, also there is mammal BMP-1/TLD associated protein enzymes different in two kinds of heredity, be appointed as mammal Toll oid sample 1 and Tolloid sample 2(" mTLL1 " and " mTLL2 ").The predomain of BMP-1/TLD sample protease must be removed by subtilisin sample proprotein convertases (SPC) proteolysis (Leighton and Kadler, j. Biol. Chem., 278:18478-18484 (2003)), to realize the complete activity of these protease.The effect of the predomain of BMP-1/TLD sample protease look like make BMP-1/TLD sample protease maintain latent form (people such as Marques, cell, 91:417-426(1997); The people such as Sieron, biochemistry, 39:3231-3239(2000); Leighton and Kadler(2003)).

The proteolysis that BMP-1/TLD related metalloproteases is responsible for being formed to extracellular matrix (ECM) relevant many extracellular proteins is ripe.These comprise multiple collagen, little be rich in leucic Dan Baiduotang proteoglycan PG, SIBLING albumen, lysyloxidase, Kallinin and from Dan Baiduotang proteoglycan PG perlecan beading element (perlecan) anti-angiogenesis (Iozzo, nat. Rev. Mol. Cell. Biol., 6: 646-656(2005); Greenspan, top. Curr. Chem., 247:149-183(2005); Ge and Greenspan(2006)).BMP-1 also relates to makes real BMP discharge from ECM, or relates to people such as activating TGF-'beta ' family member such as BMP-4, BMP-11 and GDF-8(Wolfman of hiding, proc. Natl. Acad. Sci. USA, 100:15842-15846(2003); The people such as Ge, mol. Cell. Biol., 25:5846-5858(2005)).

The BMP-1 form of initial discovery is appointed as " BMP-1-1 " (or " BMP1-1 "; SEQ ID NO:1), and other BMP-1 isotypes of being encoded by splice variant rna transcription thing are described on transcriptional level, and specify sequential suffix: BMP-1-2, BMP-1-3, BMP-1-4, BMP-1-5, BMP-1-6 and BMP-1-7.See such as, the people such as Kessler (1996); The people such as Li (1996); The people such as Wozney (1988); The people such as Janitz, j. Mol. Med., 76:141-146(1998); The people such as Takahara (1994); The people such as Hillman, genome Biol., 5(2): R8.1-R8.16(2004); And Ge and Greenspan, birth Defect Res., 78:47-68(2006).As expected, share many domains by the BMP-1 isotype of splice variant transcript encodes, comprise and guide peptide, proparea and protease (catalysis) district.In the past, only original BMP-1, i.e. BMP-1-1, protein level was confirmed, and the sequence of BMP-1-2 and other BMP-1 isotypes is drawn by the nucleotide sequence derivation of splice variant transcript, but was described on protein level.Recently, many BMP-1 isotypes have been defined as circulation in the blood of the patient with various diseases (such as chronic nephropathy and acute pancreatitis) and the blood (it is only containing BMP-1-3) of Healthy People individuality on protein level.See such as International Patent Publication No. WO 2008/011193 A2; The people such as Grgurevic, j. Am. Soc. Nephrol., 21:681-692(2011).In addition, the effect of BMP-1 in the precollagen processing of the fibrosis caused in various diseases and scar tissue, and in the patient of various diseases, comprise the discovery of blood profile of indivedual BMP-1 isotype, make BMP-1 become the target of attractive exploitation new therapy.See such as, WO 2008/011193 A2, the people (2011) such as the people such as Turtle (2004) and Grgurevic.

Although the availability of multi-medicament and operation, hundreds of thousands of them is still had to die from acute myocardial infarction every year.It is clear that, still exist and be used for the treatment of and prevent the new compositions of acute myocardial infarction and the needs of method.

summary of the invention

To the invention provides based on following discovery for the new method of acute myocardial infarction (AMI, heart attack) Diagnosis and Treat and compositions: human heart tissue contains BMP-1-4; Find that BMP-1-4 circulates in the blood of people's object with lasting AMI, but do not find in healthy individuals; And BMP-1-3 and BMP-1-4 is the therapeutic target being used for the treatment of AMI.Therefore, AMI can be diagnosed by detecting from the BMP-1-4 existence in the blood sample of people patient at present.In addition, as shown here, use the antibody for BMP-1-3 and/or the antibody for BMP-1-4 effectively reduces myocardial tissue damage degree, and even effectively promote the function regenerating heart tissue that has in the cardiac infarction district of the individuality of lasting AMI.

In method and composition described herein, BMP-1-3 protein is the BMP-1 isotype of the aminoacid sequence with SEQ ID NO:2, and BMP-1-4 protein is the BMP-1 isotype of the aminoacid sequence with SEQ ID NO:3.

In one embodiment of the invention, provide the method for the acute myocardial infarction (AMI) be used for the treatment of in people's object, it comprises the combination of using the antibody for BMP-1-3 or the antibody for BMP-1-4 or the antibody for BMP-1-3 and the antibody for BMP-1-4 to object.Preferably, antibody is neutralizing antibody.

Present invention also offers treatment individual with prevention or suppress because acute myocardial infarction is to the method for the damage of cardiac muscular tissue, it to use the combination of the antibody for BMP-1-3 or the antibody for BMP-1-4 or the antibody for BMP-1-3 and the antibody for BMP-1-4 to individuality before being included in AMI.

In another embodiment, the invention provides the method for the acute myocardial infarction in diagnosis individual human, it is included in the existence detecting in individual blood sample and have the BMP-1-4 of the aminoacid sequence of SEQ ID NO:3, or the epi-position detecting BMP-1-4 aminoacid sequence maybe can detect fragment (such as antitrypsin fragments).

The diagnostic method that the present invention is used for acute myocardial infarction advantageously uses and can carry out in conjunction with the detection thing binding molecule of BMP-1-4, and the existence instruction individuality of BMP-1-4 in the blood sample deriving from individuality has persistence acute myocardial infarction.Suitable BMP-1-4 detection thing binding molecule comprises the antibody molecule (comprising the binding fragment of polyclonal antibody and monoclonal antibody, genetic engineering antibody molecule and antibody, such as Fab fragment, F(ab') in conjunction with BMP-1-4 ₂fragment etc.) and in conjunction with fit (there is for specified protein the nucleic acid molecules of specific binding affinity) of BMP-1-4.Detect thing binding molecule for the antibody of BMP-1-4 or other BMP-1-4 to be combined (covalently or non-covalently) with providing the detectable label molecule of detectable signal, described detectable signal allows qualification by the complex of anti-BMP-1-4 antibody (or other BMP-1-4 detect thing binding molecule) and target BMP-1-4 formation for diagnosing AMI.

Further embodiment of the present invention is the method with regard to Diagnosis of Acute Myocardial Infarction and treatment individuality, and it comprises:

A () detects the existence from BMP-1-4 in the blood sample of individuality, wherein in sample, the existence instruction individuality of BMP-1-4 has persistence acute myocardial infarction;

With

B () uses the combination of the antibody for BMP-1-3, the antibody for BMP-1-4 or the antibody for BMP-1-3 and the antibody for BMP-1-4 to being detected as the individuality with persistence acute myocardial infarction in step (a).

accompanying drawing is sketched

Fig. 1 shows the domain diagram of total length (unprocessed) BMP-1-1, BMP-1-3, BMP-1-4 protein of being encoded by BMP-1 gene isotype (alternative splicing product), wherein indicates common and isotype specificity domain.Domain is not drawn to scale.The position of the corresponding splice junction in the coded sequence of each isotype is by the breach between respective egg white matter domain and bridge instruction." boot section "=signal peptide sequence.Peptide domain before " predomain "=N-terminal, it seems to make BMP-1 metalloproteases maintain latent form, and must be cut to provide completely active proteinase activity.The 3,4,3',4'-tetraketo-.beta.-carotene sample catalyst structure domain of " protease "=common.The CUB domain of " CUB "=BMP-1 isotype, wherein each CUB domain is distinguished successively by numeral.Epidermal growth factor (EGF) the spline structure territory that " EGF "=calcium combines, wherein each EGF domain is distinguished successively by numeral." ISD "=isotype specificity domain, it is for the specific C-terminal peptide domain of each BMP-1 isotype.The ISD of BMP-1-1 isotype protein is the peptide of the amino acid residue 703-730 with SEQ ID NO:1.The ISD of BMP-1-3 isotype protein is the peptide of the amino acid residue 977-986 with SEQ ID NO:2.The ISD of BMP-1-4 isotype protein is the peptide of the amino acid residue 245-302 with SEQ ID NO:3.

Fig. 2 shows the figure of MB creatine kinase protein (creatine kinase myocardial band protein) (" CK-MB ") level (units per liter, " U/L ") compared with the time (" my god ") after the left coronary artery surgical ligation of inducing AMI in acute myocardial infarction (AMI) rat blood with ligation induction.Solid diamond=with the monoclonal antibody (" BMP1-3 mAb ") for BMP-1-3 process have ligation induction AMI rat blood in CK-MB level.Open squares=not by the CK-MB level (contrast) had in the control rats blood of the AMI of ligation induction of BMP1-3 mAb therapy processes.Asterisk instruction compared with that in control rats, with the significant difference (* p<0.05) in the CK-MB level in the rat of antibody treatment.About details see Example 4.

Fig. 3 shows the figure of MB creatine kinase protein (" CK-MB ") level (units per liter, " U/L ") compared with the time (" my god ") after the left coronary artery surgical ligation of inducing AMI in acute myocardial infarction (AMI) rat blood with ligation induction.Solid diamond=with the polyclonal antibody (" BMP1-4 Ab ") for BMP-1-4 process have ligation induction AMI rat blood in CK-MB level.Open squares=not by the CK-MB level (contrast) had in the control rats blood of the AMI of ligation induction of BMP1-4 Ab therapy processes.Asterisk instruction compared with that in control rats, with the significant difference (* p<0.05) in the CK-MB level in the rat of antibody treatment.About details see Example 5.

Fig. 4 show have ligation induction acute myocardial infarction (AMI) rat blood in troponin t protein level (μ g/L) with induce AMI left coronary artery surgical ligation after time (my god) compared with figure.The troponin t level had in the rat blood of the AMI of ligation induction of solid diamond=process with the combination (" BMP1-3 mAb+BMP1-4 mAb ") of the monoclonal antibody for BMP-1-3 and the monoclonal antibody for BMP-1-4.Open squares=not by the troponin t level (contrast) had in the control rats blood of the AMI of ligation induction of antibody treatment.Asterisk instruction compared with that in control rats, with the significant difference (* p<0.05) in the troponin t level in the rat of antibody treatment.About details see Example 6.

Fig. 5 shows for the rat (" BMP1-3 mAb ") with BMP-1-3 mAb process with not by the control rats (" contrast ") with AMI of antibody treatment, before surgery (preoperative), after the ligation operation of inducing AMI when one week (" 1 week ") and when the Post operation one month of induction AMI (" 1 month "), the reconstruct PET scanogram of rat heart.Arrow instruction for the rat with BMP1-3 mAb process, after surgery one week and one month time defect area.In the animal with BMP-1-3 mAb process, clearly indicate the recovery of the function cardiac muscular tissue in the original infarcted region of heart after one month, and in the heart of undressed control animal, the forfeiture of functional organization is still after one month apparent.About details see Example 8.

Fig. 6 show after coronary artery ligation use or do not use BMP-1-3 monoclonal antibody therapy, from the microphotograph of the histologic analysis of the cardiac muscle in rat.Fig. 6 A shows when there is not antibody therapy, after the left coronary artery ligation of inducing acute myocardial infarction (AMI) when one week, from the heart sections (amplification 4X) of the infarcted region of rat heart.Rectangle in Fig. 6 A amplifies at Fig. 6 B.Fig. 6 B shows sirius red dyeing (with 20X amplification) of the tissue of the rectangular area in Fig. 6 A, indicates early stage collagen deposition.See the arrow in Fig. 6 B.Fig. 6 C shows with h and E dyeing, from the Cardiac muscle sections of undressed rat with AMI, disclose after one month by the cardiac muscle fiber damaged around residual fibers scar tissue.See the arrow in Fig. 6 C.Fig. 6 D shows the heart sections in the cardiac infarction district from rat, described rat before the left coronary artery ligation of induction AMI with BMP-1-3 mAb(15 μ g/kg) process, and subsequently between period 1 after surgery every day with BMP-1-3 mAb process.Fibrosis region after AMI is significantly less than observe in control rats that.See the arrow in Fig. 6 D.Fig. 6 E shows the more magnification at high multiple in the region by the arrow instruction in Fig. 6 D, discloses new regenerated muscle fibers speckle.See the arrow in Fig. 6 E.Fig. 6 F shows the more detailed view in the region by the instruction of arrow in Fig. 6 D, discloses the cell around the muscle fiber and fibrous tissue that are recently formed, its with observe in control rats that compared with more not fine and close.About details see Example 9.

detailed Description Of The Invention

Described hereinly the present invention is based on following discovery, BMP-1-3 and BMP-1-4 protein is all present in and has persistence acute myocardial infarction (" AMI ", " heart attack ") adult individu blood in, and these two kinds of BMP-1 isotypes are also the therapeutic target being used for the treatment of AMI.

In order to make the present invention be fully understood, define following term.

The aminoacid sequence of total length described herein (unprocessed) BMP-1-1 protein has following aminoacid sequence:

。

The aminoacid sequence of total length described herein (unprocessed) BMP-1-3 protein has following aminoacid sequence:

。

The aminoacid sequence of total length described herein (unprocessed) BMP-1-4 protein has following aminoacid sequence:

。

Unless otherwise stated, when term " about " and " approximately " and amount, digital or value combinationally uses time, then the combination describes independent described amount, numeral or value and add or deduct the amount of this amount, numeral or value 10%, numeral or value.As non-limitative example, phrase " about 40% " and " about 40% " disclose " 40% " and " from 36% to 44%, comprising end value ".

As used herein with understand, " antibody " or " antibody molecule " refers to specific binding members, it is no matter be natural generation, or the protein molecule of synthesis or semi-synthetic generation or its part, there is antigen-binding domains, and in conjunction with certain target molecules (antigen), described antigen-binding domains comprises immunoglobulin light chain variable region or domain (V _l) or its part, immunoglobulin heavy chain variable region or domain (V _h) or its a part of or its combination.Any polypeptide or protein molecule with antigen-binding domains also contained in term " antibody ", described antigen-binding domains or homology identical with the antigen-binding domains of immunoglobulin.Antibody can be " polyclone ", namely, the colony of antigen binding molecules produces in multiple different cell, and the different loci thus on antigen is combined, or " monoclonal ", that is, the colony of the same antigen binding molecule produced by individual cells system, it combines with the only site (i.e. the identical epi-position of antigen) on antigen.As used herein with to understand, the example of antibody molecule comprises any one in immunoglobulin (such as, IgG, IgM, IgA, IgE, IgD) kind of well-known kind and isotype thereof; Comprise the immunoglobulin fragment of antigen-binding domains, such as Fab or F(ab') ₂molecule; Single-chain antibody (scFv) molecule; Dual scFv molecule; Single domain antibody (dAb) molecule, it has the function antigen-binding domains of three CDR only comprising single heavy-chain variable domains, its can to combine to antigen with the ratio of 1:1 and without the need to corresponding light variable domains (see people such as such as Ward, nature, 341:544-546(1989); International publication number WO 90/05144; The people such as Hamers-Casterman, nature, 363:446-448(1993), the people such as Muyldermans, protein Eng., 7:1129-1135(1994)); FD molecule (being made up of the antibody VH district be connected with heavy chain of antibody constant domain CH1, CH2, CH3 and optional CH4 district); Homodimer; And comprise the fusion rotein of this quasi-molecule.Binary consists of the combination of two binary monomers, and it forms the dimer containing two complete antigen-binding domains, wherein each binding structural domain itself by the intermolecular combination from two monomer regions separately formed (see people such as such as Holliger, proc. Natl. Acad. Sci. USA, 90: 6444-6448(1993)).In compositions described herein and method, the useful antibody for BMP-1-3 or BMP-1-4 can be also bi-specific antibody, and it comprises another antigen-binding domains of the antigen-binding domains of the molecule of specific binding BMP-1-3 and the molecule of specific binding BMP-1-4.The antibody molecule for BMP-1-3 or BMP-1-4 that may be used in compositions described herein and method also can be that dual variable domains (DVD) associated proteins is (see such as, International Patent Publication No. WO 2007/024715), it comprises the antigen-binding domains of specific binding BMP-1-3, or the antigen-binding domains of specific binding BMP-1-4, or comprise the antigen-binding domains of molecule of specific binding BMP-1-3 and another antigen-binding domains of the molecule of specific binding BMP-1-4.Above-mentioned all molecules are associated proteins useful in method described herein, because they comprise the function binding structural domain about BMP-1-3 and/or the function binding structural domain about BMP-1-4.The antibody be combined with BMP-1-3 or BMP-1-4 is alternately called as " BMP-1-3 antibody " or " BMP-1-4 antibody " in this article respectively, and is called as respectively " anti-BMP-1-3 antibody " and " anti-BMP-1-4 antibody ".

" antibody of separation " means to be substantially free of the antibody of other antibody molecules and the antibody fragment with different antigenic specificity (such as, the antibody of the separation of specific binding particular B MP-1 isotype such as BMP-1-3 or BMP-1-4, is substantially free of the antibody molecule of the antigen of specific binding except particular B MP-1 isotype).But " antibody of separation " of specific binding particular B MP-1-3 such as can have cross reactivity from the BMP-1-3 of other species with other antigens.In addition, the antibody of separation can be substantially free of other cell materials and/or chemicals.

Term " monoclonal antibody " or " mAb " refer to the antibody of the antibody molecule populations deriving from homogeneity substantially, and the individual antibody molecule that namely colony comprises is identical, except can with except the naturally occurring sudden change existed on a small quantity.Monoclonal antibody is high degree of specificity, for single antigen.In addition, formed from the polyclonal antibody preparations of the different antibodies molecule of the different antigenic determinants (epi-position) generally included for antigen and contrast, each mAb molecule is for the single epi-position of antigen.Qualifier " monoclonal " should not be construed as needs the antibody by any ad hoc approach to produce.

Term " people's antibody " comprises and has the variable region of derived from human germ-line immunoglobulin sequence and the antibody of constant region.People's antibody can comprise such as at CDR and particularly in CDR-H3, can't help the amino acid residue (such as, by external random or site-specific mutagenesis or the sudden change introduced by the somatic mutation in body) of human germline immunoglobulin's sequential coding.But term " people's antibody " does not comprise the antibody be wherein transplanted to derived from the CDR sequence of another mammalian species germline such as mice on people's frame sequence.

Term " recombinant human antibody " comprises by recombination method preparation, everyone antibody of expressing, producing or being separated, such as use the antibody that the recombinant expression carrier be transfected in host cell is expressed, antibody (the Hoogenboom be separated from the combination people antibody library of restructuring trends Biotechnol., 15:62-70(1997); Azzazy and Highsmith, clin. Biochem., 35:425-445(2002); Gavilondo and Larrick, bioTechniques, 29:128-145(2000); Hoogenboom and Chames, immunol. Today, 21:371-378(2000)), from for human immunoglobulin gene be genetically modified animal (such as mice) be separated antibody (see people such as such as Taylor, nucl. Acids Res., 20:6287-6295(1992); Kellermann and Green, curr. Opin. Biotechnol.,13:593-597(2002); The people such as Little, immunol. Today, 21:364-370(2000)); Or the antibody prepared by any other method, express, produce or be separated, any other method described relates to the montage of human immunoglobulin gene's sequence and other DNA sequence.This type of recombinant human antibody has variable region and the constant region of derived from human germ-line immunoglobulin sequence.In certain embodiments, but, to this type of recombinant human antibody implement in vitro mutagenesis (or, when using animal genetically modified for people Ig sequence, somatic mutagenesis in body), and therefore, the aminoacid sequence in VH and the VL district of recombinant antibodies is such sequence, although its derived from and relate to people germline VH and VL sequence, may and non-natural to be present in body in people's antibody germline bank.

Term " chimeric antibody " refers to the antibody of heavy chain and light-chain variable sequence and the constant-region sequences from another species comprised from species, such as, have the antibody of Mus heavy chain and the variable region of light chain be connected with human constant region.

Term " CDR " refers to the complementary determining region in antibody variable region.There are three CDR in each antibody variable region, and be appointed as " CDR1 ", " CDR2 " and " CDR3 ", wherein by the routine agreement as adopted herein, " CDR1 " refers to most N-terminal near-end in three CDR in antibody variable region, and most C-terminal near-end in " CDR3 " refers in antibody variable region three CDR.CDR in antibody heavy chain variable region (VH) is appointed as " CDR-H1 ", " CDR-H2 " and " CDR-H3 ", and the CDR in antibody chain variable region (VL) is appointed as " CDR L1 ", " CDR-L2 " and " CDR-L3 ".

Term as used herein " CDR group " refers to one group of three CDR existed in single variable region, and it can the defined epitope of conjugated antigen molecule.These CDR really trimming circle limit according to different numbering systems difference.The system that described by Kabat (people such as Kabat, sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Maryland(1987) and (1991)); The people such as Kabat, sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH publication number 91-3242(1991)) be the most widely used numbering system.Kabat numbering system provides the residue numbering system for the residue in variable region, and provides the exact residue border of restriction three CDR.Devised other numbering systems afterwards, but Kabat numbering system is still the resi-dues be used to specify in antibody variable region, and the most widely used numbering system of aminoacid sequence for the identification of each CDR in antibody variable region.

In the past between Two decades years, the growth of the extensive public database of the aminoacid sequence in variable heavy chain and light chain district and analysis, caused the understanding to the typical boundary between the framework region (FR) in variable region sequences and CDR sequence, and make that those skilled in the art can number according to Kabat, Chothia numbering or other system accurately determine CDR.See such as, Martin, " Protein Sequence and Structure Analysis of Antibody Variable Domains, " the 31st chapter, in antibody Engineering, (Kontermann and D ü bel, editor) (Springer-Verlag, Berlin, 2001), particularly 432-433 page.Provided hereinafter the process useful of the aminoacid sequence of the Kabat CDR determined in the aminoacid sequence in variable heavy chain (VH) and variable light (VL) district:

In order to identify CDR-L1 aminoacid sequence:

From about 24 aminoacid of the aminoterminal in VL district;

Residue before CDR-L1 sequence is always cysteine (C);

Residue after CDR-L1 sequence is always tryptophan (W) residue, is generally Trp-Tyr-Gln(W-Y-Q), also can be Trp-Leu-Gln(W-L-Q), Trp-Phe-Gln(W-F-Q) and Trp-Tyr-Leu(W-Y-L);

Length is generally 10-17 amino acid residue.

In order to identify CDR-L2 aminoacid sequence:

All the time after the end of CDR-L1,16 residues start;

Residue before CDR-L2 sequence is generally Ile-Tyr(I-Y), also can be Val-Tyr(V-Y), Ile-Lys(I-K) and Ile-Phe(I-F);

Length is always 7 amino acid residues.

In order to identify CDR-L3 aminoacid sequence:

All the time after the end of CDR-L2,33 residues start;

Residue before CDR-L3 aminoacid sequence is always cysteine (C);

Residue after CDR-L3 sequence is always Phe-Gly-X-Gly(F-G-X-G) (SEQ ID NO:4), wherein X is arbitrary amino acid;

Length is generally 7-11 amino acid residue.

In order to identify CDR-H1 aminoacid sequence:

From about 31 amino acid residues of the amino terminal in VH district and all the time 9 residues after cysteine (C) start;

Residue before CDR-H1 sequence is always Cys-X-X-X-X-X-X-X-X(SEQ ID NO:5), wherein X is arbitrary amino acid;

Residue after CDR-H1 sequence is always Trp(W), be generally Trp-Val(W-V), also can be Trp-Ile(W-I) and Trp-Ala(W-A);

Length is generally 5-7 amino acid residue.

In order to identify CDR-H2 aminoacid sequence:

All the time after the end of CDR-H1,15 aminoacid start;

Residue before CDR-H2 sequence is generally Leu-Glu-Trp-Ile-Gly(L-E-W-I-G) (SEQ ID NO:6), but also there is other changes;

Residue after CDR-H2 sequence is generally Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala(K/R-L/I/ V/F/T/A-T/S/I/A);

Length is generally 16-19 amino acid residue.

In order to identify CDR-H3 aminoacid sequence:

All the time after the end of CDR-H2,33 aminoacid start and start at rear 3 residues of cysteine (C) all the time;

Residue before CDR-H3 sequence is always Cys-X-X(C-X-X), wherein X is arbitrary amino acid, is generally Cys-Ala-Arg(C-A-R);

Residue after CDR-H3 sequence is always Trp-Gly-X-Gly(W-G-X-G) (SEQ ID NO:7), wherein X is arbitrary amino acid;

Length is generally 3-25 amino acid residue.

Term " CDR grafted antibody " refers to such antibody molecule, it comprises heavy chain from species and light-chain variable sequence, but the sequence in the one or more CDR districts wherein in VH and/or VL district replaces with the CDR sequence of another species, such as there is the antibody of people's heavy chain and variable region of light chain, wherein one or more people CDR(such as, CDR3) replaced with Mus CDR sequence.The method be transplanted to by the CDR of the antibody of species in the variable domains of the antibody of another species is well-known in the art.See such as, the people such as Jones, nature, 321:522-525(1986); The people such as Riechmann, nature, 332:323-327(1988); The people such as Verhoeyen, science, 239:1534-1536(1988); With people such as Queen, proc. Natl. Acad. Sci. USA, 86:10029-10033(1989).

" circulation " and " circulation " describes anything of to be advanced by the vascular system of individuality or otherwise transporting.

Term " disease " and " disease " are synonyms, and refer to any pathological condition, have nothing to do with reason or cause of disease agent." defect " in tissue refers to the position of exception or defect(ive) structure growth.The feature of " disease " or " disease " can be one or more " defects " in one or more tissues.Object disease (or disease) of the present invention is acute myocardial infarction (" AMI ", " heart attack ").

As used herein, term " therapy " and " treatment " refer to and palliate a disease or one or more symptoms of disease or any scheme of performance, it suppresses the process of disease or disease, stagnate disease or the progress of disease or the progress (cause and disappear) of reverse disease or disease, or prevent disease or the outbreak of disease.Term " therapy " comprises one or more symptoms or the performance of prevention (preventing) disease, comprises the degree improved or suppress symptom or performance, and when there is not treatment, described symptom or performance otherwise will characterize disease.

" treatment effective dose " is such compound (such as the antibody of BMP-1-3, for the antibody of BMP-1-4 or its combination) amount, and it suppresses the progress of disease wholly or in part; One or more symptoms palliated a disease at least partly; Or strengthen or promote to be used for the treatment of the response to treatment of the another kind of compound of disease or effect favourable in other respects.Treatment effective dose also can be the effective amount of prevention.The effective amount for the treatment of will depend on the size of patient and sex, disease to be treated, the seriousness of disease and the result of seeking.For given individual human, treatment effective dose can be determined by method known to those skilled in the art.

When for describing multiple proteins disclosed herein or polypeptide, term " separation " means protein or polypeptide is identified, and with the Component seperation of its natural surroundings and/or recovery.The contaminant component of its natural surroundings usually disturbs the diagnosis of this polypeptide or the material of therapeutic use, and can comprise enzyme, hormone and other protein or nonprotein kind.The protein be separated or polypeptide are included in through transforming the protein or polypeptide of expressing original position in its reconstitution cell as, because at least one component of the natural surroundings of this protein or polypeptide will not exist.But usually, the protein of separation or polypeptide will be prepared by least one purification step.

The compositions or the method that are described as " comprising " one or more element of specifying or step are herein open, mean this element of specifying or step is required, but other elements or step can add in the scope of said composition or method.In order to avoid tediously long, will also be understood that be described as herein " comprising " (or " it comprises ") one or more element of specifying or any compositions of step or method also describe compositions or the method for corresponding more restricted " being substantially made up of the identical element of specifying or step " (or " it is made up of the identical element of specifying or step substantially "), mean said composition or method comprises the required element or step of specifying, and other element or the step of the fundamental sum novel feature not affecting in fact said composition or method can be comprised.Will also be understood that any compositions of being described as " comprising " one or more element of specifying or step or " being substantially made up of one or more element of specifying or step " herein or method also describe corresponding more restricted and the compositions of enclosed " being made up of the element of specifying or step " (or " it is made up of one or more element of specifying or step ") or method, to get rid of any other unspecified element or step.In any compositions disclosed herein or method, the known or open equivalent of any required element of specifying or step can replace this element or step.

Unless otherwise stated, the implication of other terms and those skilled in the art understand with use identical, described field comprises medical science, immunology, biochemistry, molecular biology and tissue regeneration field.

The present invention is based on following discovery: BMP-1-4 is present in the blood of the individual human with persistence acute myocardial infarction (AMI), but is not present in the blood of healthy individuals.Be present in the BMP-1-3 isotype protein in the blood circulation of healthy individuals, be also present in the blood of the individuality with persistence AMI.Therefore, BMP-1-4 can be used as the blood biological labelling (biomarker) of new AMI.

As previously shown, by analyzing blood for the existence of one or more peptides (such as, tryptic peptide) of particular B MP-1 isotype uniqueness, the BMP-1 isotype protein in blood sample can be detected.See such as, WO 2008/011193; The people such as Grgurevic (2011).As shown in Examples below 1, this kind of peptide analysis confirms the existence of BMP-1-4 isotype protein on protein level in people first.In addition, BMP-1-4 protein in the blood of patient with persistence acute myocardial infarction, instead of detects in the blood of healthy volunteer.Use for the antibody of BMP-1-4 and BMP1-3, BMP-1-4 and BMP1-3 be positioned developmental Human embryo heart and have persistence AMI individual human heart sections in (data do not show).Therefore, BMP-1-4 expresses usually in normal cardiac tissue, but appears in the blood of the individuality with persistence AMI.The appearance of BMP-1-4 in the blood of individual human with persistence AMI also with blood plasma troponin t(" Tn-T ") appearance to rise with MB creatine kinase (CK-MB) level and associate.Therefore, BMP-1-4 can be used as the blood biomarker of AMI.

BMP-1-4 described herein appears in the blood of the individuality with persistence AMI and BMP-1-4 is positioned the discovery in healthy heart tissue, and instruction BMP-1-1 and BMP-1-3 promotes that the previous discovery of the fibrosis in other organs and scar tissue is (see such as, the people such as Turtle (2004), WO 2008/011193, the people such as Grgurevic (2011)), cause the present inventor to study arbitrary in BMP-1-3 and BMP-1-4 or whether both can be used as the therapeutic target for the treatment of AMI.The result of study being used in the AMI normal rat model described in Examples below clearly shows, BMP-1-3 and BMP-1-4 is the therapeutic target being used for the treatment of AMI.Such as, compared with that in the undressed control rats with AMI, significantly lower biomarker CK-MB blood plasma level is caused to rise to the monoclonal antibody (" BMP-1-3 mAb ") that the rat with AMI is used for BMP-1-3.See embodiment 4 and Fig. 2.Compared with that in the undressed control rats with AMI, significantly lower CK-MB blood plasma level is also caused to rise to the antibody that the rat with AMI is used for BMP-1-4.See embodiment 5 and Fig. 3.Compared with undressed control rats, the combination of the rat with AMI being used to BMP-1-3 mAb and BMP-1-4 mAb also causes remarkable lower level troponin t(" Tn-T ") blood plasma level.See embodiment 6 and Fig. 4.

In addition, the therapeutic effect rat with AMI being used to BMP-1-3 mAb is also indicated by the echocardiography of cardiac dimensions and function, compared with those in undressed control rats, described echocardiography discloses significantly higher relaxing period (IVSd) and systole (IVSs) interventricular septum size, significantly lower left ventricular internal dimension systole (LVIDs), significantly lower left ventricular contraction after date wall thickness (LVPWs), significantly higher ejection fraction (EF) and remarkable higher Fractional shortening (ES).In fact, the cardiac dimensions of the rat of antibody treatment and function class are similar to without those of the sham-operation rat of AMI.See embodiment 7 and table 1.

Also show and to promote after AMI higher-quality scar tissue and function cardiac muscular tissue in cardiac infarction district with BMP-1-3 mAb treatment.As described in example 8 above, use positron emission tomography (PET), by through a month process monitoring cardiac infarction district, greatly show this curative effect of Antybody therapy.As shown in Figure 5, AMI operation induction before (" preoperative "), after surgery one week time (" 1 week ") and after surgery one month time (" 1 month "), the heart with the rat of AMI carries out PET scanning.Clearly show with the rat heart of BMP-1-3 mAb process and the PET scanogram of undressed control rats: after surgery one week time, nonfunctional tissue in infarcted region, although the infarcted region of undressed control rats seems than processing that more remarkable of rat.See the cardiac image in Fig. 5 when " 1 week ".But induce latter one month in the operation of AMI, PET scanogram is disclosed in the greatest differences of the liver mass generated in the primitive cardiac infarcted region of process and undressed animal.Especially, the PET scanogram accepting the rat heart that BMP-1-3 mAb treats is presented at a large amount of recoveries of the function cardiac muscular tissue in infarcted region, and the nonfunctional in the infarcted region of undressed control rats is organized and is clearly retained, and even than more remarkable one week time.See the cardiac image in Fig. 5 when " January ".Result shows, and compared with the repair tissue generated in the heart of undressed control rats, the repair tissue accepted in the rat heart of antibody therapy clearly has more high-quality and greater functionality.

As described in Examples below 9, from have AMI undressed control rats heart and carry out the histologic analysis with the cardiac muscular tissue of the rat of AMI of personal BMP-1-3 mAb process, also show the favourable effect of use BMP-1-3 antibody treatment.Especially, after the left coronary artery surgical ligation of inducing AMI when one week, the sirius red dyeing from the cardiac muscular tissue of undressed control rats discloses early stage collagen deposition (see Fig. 6 B).After surgery one month time, from the cardiac muscular tissue of undressed control rats sirius red dyeing disclose by the cardiac muscle fiber damaged clearly around residual fibers scar tissue (see Fig. 6 C).By contrast, with undressed control rats that compared with, the fibrosis area after AMI when one week is obvious less (see Fig. 6 D) in the cardiac muscular tissue of the rat with BMP-1-3 mAb process.The more magnification at high multiple of the tissue shown in Fig. 6 D disclose muscle fiber speckle (see Fig. 6 E) and the surrounding recently formed cell and than that obviously more unsound fibrous tissue (see Fig. 6 F) observed in undressed control rats.

Any one in multiple method known in the art may be used for producing polyclone or monoclonal antibody molecule, its specific binding specific purpose BMP-1 isotype (such as BMP-1-3 or BMP-1-4), or comprise the part of specific purpose BMP-1 isotype of at least one epi-position (that is, specific antigen binding site) for particular B MP-1 isotype.

Polyclonal antibody can use standard method known in the art to produce, wherein under the condition causing immunne response by animal, by antigen (such as, BMP-1-3, BMP-1-4 or comprise the peptide of epi-position of BMP-1-3 or BMP-1-4) be applied to animal, cause producing the antibody for this antigen.Usually, this type of polyclonal antibody produces in the blood of animal, and can be separated in the sera components of blood (antiserum).Be further purified and the polyclonal antibody preparations strengthening purity or the antibody being separated particular types from antiserum can be provided.

For the monoclonal antibody (mAb) that the preferred antibody molecule in compositions described herein and method is for BMP-1-3 and BMP-1-4.Monoclonal antibody can use the obtainable Standard hybridoma technology in this area to be prepared.This technology describes in the standard laboratory manual of this area.See such as, the people such as Harlowe, antibodies:A Laboratory Manual, the second edition(Cold Spring Harbor Laboratory Press, 1988); monoclonal Antibodies and T-Cell Hybridomas(Elsevier, New York, 1981); Be incorporated to by reference herein.Use any one in the obtainable multiple additive method in this area, the antibody for BMP-1-3 and BMP-1-4 can be generated.Such as, use selectivity lymphocyte antibody method (SLAM), can by the antibody of single separation lymphocyte generation for BMP-1-3 and BMP-1-4.See such as, U.S. Patent number 5,627,052; International publication number WO 92/02551; The people such as Babcook, proc. Natl. Acad. Sci. USA, 93:7843-7848(1996); Be incorporated to by reference herein.All or part of the transgenic animal comprising human immunoglobulin gene's seat can also be used, prepare the antibody for BMP-1-3 and BMP-1-4, when this transgenic animal BMP-1-3 or BMP-1-4 protein or its fragments of peptides immunity inoculation, described human immunoglobulin gene's seat will produce people's antibody.See such as, the people such as Green, nature Genetics, 7:13-21(1994); U.S. Patent number 5,916,771; International Patent Publication No. WO 91/10741; Be incorporated to by reference herein.Additive method for generation of BMP-1-3 and the BMP-1-4 antibody that can be used in compositions described herein and method includes but not limited to, phage display (such as, be incorporated to the people such as Brinkmann herein by reference, j. Immunol. Methods, 182:41-50(1995); The people such as Ames, j. Immunol. Methods, 184:177-186(1995), the people such as Kettleborough, eur. J. Immunol., 24:952-958(1994))), yeast display method is (see such as, be incorporated to U.S. Patent number 6,699,658 herein by reference), express with the antibody library as RNA-protein fusions (see such as, being incorporated to International Patent Publication No. WO 98/31700 herein by reference).

Preferably, use the peptide antigen with the aminoacid sequence (the amino acid residue 972-986 of SEQ ID NO:2) of R-Y-T-S-T-K-F-Q-D-T-L-H-S-R-K, produce BMP-1-3 mAb.Particularly preferred BMP-1-3 mAb is called _ _ _ _, produced by hybridoma cell line, described hybridoma cell line passes through ProMab(Richmond, California, the U.S.) order preparation, and according to budapest treaty on April 24th, 2013 be preserved in German Culture Collection (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikrooganismen und Zellkulturen GmbH) (" DSMZ ") (registration number _ _ _ _ _ _).

Preferably, use the peptide based immunogens with the aminoacid sequence (SEQ ID NO:8) of C-G-S-R-N-G-A-S-F-P-S-S-L-E-S-S-T-H-Q-A, produce BMP-1-4 mAb.BMP-1-4 mAb, be called _ _ _ _ _, by ProMab(Richmond, California, USA) order and produce.

The rodent hybridoma producing monoclonal antibody (" mAb ") is the constant region of coding mAb molecule and the ready-made DNA source of variable region.What be particularly useful is indivedual complementary determining regions (" CDR ") of encoding BMP-1-3 mAb or BMP-1-4 mAb and being separated and sequencing of the DNA of framework region (" FR ").Coding indivedual CDR, FR of rodent BMP-1-3 mAb or BMP-1-4 mAb and/or the isolated or synthesized DNA of its part, can in the standard method easily for the production of other recombinant antibody molecules multiple in conjunction with BMP-1-3 or BMP-1-4.This type of recombinant antibody molecule includes but not limited to, CDR grafted antibody molecule; Chimeric antibody, humanized antibody; The humanized antibody of Affinity maturation; Single-chain antibody (" scFv ") molecule; Dual scFv molecule; Homodimer; In conjunction with arbitrary in BMP-1-3 or BMP-1-4 or both bi-specific antibodys; And in conjunction with arbitrary in BMP-1-3 and BMP-1-4 or both dual variable domain immunoglobin associated proteins.Particularly preferred recombinant antibodies is humanized antibody, and it combines the antigen (BMP-1-3 or BMP-1-4) identical with original rodent mAb, but when being expelled in people, has little immunogenicity.See such as U.S. Patent number 5,693,762; The people such as Queen (1989); European patent number 0 239 400 B1.

Preferably, the antibody for BMP-1-3 and BMP-1-4 used in the method and composition of the present invention being used for the treatment of acute myocardial infarction is neutralizing antibody, as by following confirmation: the ability of the precollagen cutting that this antibody suppresses BMP-1-3 or BMP-1-4 to mediate in vitro (see such as, the people such as Kessler (1996); The people such as Li (1996); The people such as Garrigue-Antar, j. Biol. Chem., 276(28): 26237-26242(2001); The people such as Hartigan, j. Biol. Chem., 278(20): 18045-18049(2003)); Ability that the dentin extracellular matrix (DMP-1) that this antibody suppresses BMP-1-3 or BMP-1-4 to mediate in vitro cuts (see such as, the people such as Qin, j. Biol. Chem., 278(36): 34700-34708(2003); The people such as Steiglitz, j. Biol. Chem., 279(2): 980-986(2004)); Or this antibody suppress in the rat model of acute myocardial infarction the degree of injury to cardiac muscular tissue ability (see people such as Zornoff, arq. Bras. Cardiol., 93(3): the rat model summary 403-408(2009)).

The method being used for the treatment of individual acute myocardial infarction (AMI) according to the present invention comprises the step of the combination of using the antibody for BMP-1-3, the antibody for BMP-1-4 or the antibody for BMP-1-3 and the antibody for BMP-1-4 to individuality.Preferably, the antibody molecule being used for the treatment of individual human AMI has such region and domain, its behaviour antibody those or be those of people's antibody substantially so that reduce use this antibody with the individuality for the treatment of AMI in cause the probability of immunne response.Therefore, the antibody for BMP-1-3 and BMP-1-4 being used for the treatment of AMI is preferably human antibody or humanized antibody.More not preferably, this antibody is chimeric antibody.More not preferably, this antibody lacks any domain of derived from human antibody or the non-human antibody in region.

For treating the use in the acute myocardial infarction (AMI) in individual human according to the present invention, use well-known in the art for the preparation of technology and the composition for treatment antibody being applied to the pharmaceutical composition of individual human, preparation comprises the compositions of the combination of the antibody molecule for BMP-1-3 or the antibody molecule for BMP-1-4 or two kinds of antibody molecules.The compositions comprising the combination of the antibody for BMP-1-3 or the antibody for BMP-1-4 or two kinds of antibody molecules can be prepared for being used by any one in multiple route of administration or mode.The compositions comprising the combination of the antibody for BMP-1-3 or the antibody for BMP-1-4 or two kinds of antibody molecules can be prepared for using outside parenteral or parenteral.Preferably, the compositions preparation comprising the combination of the antibody for BMP-1-3 or the antibody for BMP-1-4 or two kinds of antibody molecules being used for the treatment of AMI for parenteral administration, such as but not limited to intravenous, subcutaneous, intraperitoneal or intramuscular administration.More preferably, compositions preparation is used for intravenous.This type of parenteral administration is carried out preferably by the injection of compositions or infusion.

For being applied to the compositions comprising the combination of the antibody for BMP-1-3 or the antibody for BMP-1-4 or two kinds of antibody molecules of individual human, arbitrary or two kinds of antibody molecules of the effective dose combined with the acceptable component of one or more pharmacy such as pharmaceutically acceptable carrier (vehicle, buffer), excipient or other compositions can be comprised." pharmacy is acceptable " means the physiological compatible of compound, component or composition in compositions and individual human, and do not damage the effective active of BMP-1-3 antibody or BMP-1-4 antibody component, or do not damage desirable characteristics or the activity of other components that may exist in the compositions of individual human to be administered.The example of pharmaceutically acceptable carrier includes but not limited to, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol etc. and combination thereof.In many cases, preferably include isotonic agent, include but not limited to sugar; Polyhydric alcohols is as mannitol or sorbitol; Sodium chloride; And combination.Pharmaceutically acceptable carrier can comprise a small amount of auxiliary substance further, such as wetting agent or emulsifying agent, antiseptic or buffer agent, to strengthen shelf life or the effectiveness of said composition.Excipient is generally provide any compound of required feature or the combination of compound to compositions.PH value in compositions can adjust as required, such as to promote or to maintain the solubility of composition component, maintain the stability of one or more compositions in preparation, and/or prevent the upper potential undesirable growth of microorganism that may introduce of some points in operation.

Comprise the compositions of the combination of BMP-1-3 antibody or BMP-1-4 antibody or two kinds of antibody molecules, also one or more other compositions can be comprised, such as other pharmaceutical agents (such as, antibiotic, anti-inflammatory compound, antiviral agent, anticarcinogen), filler, formulation adjuvant and combination thereof.

Various ways can be taked according to compositions of the present invention.These include but not limited to, liquid, semisolid and solid dosage forms, dispersion, suspension, tablet, pill, powder, liposome and suppository.Preferred form depends on expection route of administration.Preferred composition takes injection or can the form of infusion solution, such as, be similar to those the compositions (such as, as being used for the treatment of property TNF-Alpha antibodies molecule adalimumab or infliximab) used approved and use for the treatment antibody in people.In preferred embodiments, BMP-1-3 antibody or BMP-1-4 antibody or two kinds of antibody molecules be combined through intravenous injection or infusion is used.In another embodiment, antibody is used by intramuscular or subcutaneous injection.

Therapeutic combination must be aseptic and stable under manufacture and storage requirement.Said composition can be mixed with solution, microemulsion, dispersion, liposome or other be suitable for the structure of high drug level.Sterile injectable solution can be prepared by following: mix in appropriate solvent by reactive compound (namely for the antibody of BMP-1-3 or for the antibody of BMP-1-4 or the combination of two kinds of antibody molecules) with aequum, optionally together with to one of compositions composition providing favorable characteristics or combination, when needing, be filtration sterilization subsequently.Usually, dispersion is prepared by following: mixed by active component in sterile carrier, described sterile carrier contains basic dispersion medium (such as, sterilized water, sterile isotonic saline etc.), and may be optionally one or more other compositions enough needed for dispersion.When the sterile lyophilized powder for the preparation of sterile injectable solution, preferred preparation method comprises vacuum drying and spraying dry, this by the solution of its previous aseptic filtration produce active component add any in addition needed for the powder of composition.The adequate liquidity of solution can be maintained by following: such as, uses coating such as lecithin, maintains required particle size in the case of a dispersion, and uses surfactant.The prolongation that can realize Injectable composition by comprising the reagent (such as Monostearate and/or gelatin) postponing to absorb in the composition absorbs.

The combination of the antibody for BMP-1-3 or the antibody for BMP-1-4 or two kinds of antibody molecules can be used by multiple method known in the art, although preferred route of administration or mode are parenteral administration, and are more preferably intravenous and use.As should be understood as technical staff, route of administration or mode will change according to results needed.In some embodiment, antibody can avoid being prepared together with the quick carrier discharged with protection compound, and such as Co ntrolled release preparation, comprises implants, transdermal skin patches and microencapsulated delivery system.Biodegradable and biocompatible polymer can be used, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, poe and polylactic acid.The multiple method preparing this type of preparation is well known by persons skilled in the art.

Antibody molecule in conjunction with BMP-1-3 or BMP-1-4 may be used for any one in the obtainable multiple immune detection system based on antibody in this area and form, for detecting required antigen in vitro or in body.This type systematic and form are easily applicable to BMP-1-3 or BMP-1-4 in any one detecting or measure in multiple combination thing, and described compositions includes but not limited to, whole blood, blood plasma, serum, Various Tissues extract and body fluid.Be applicable to detect this type systematic of BMP-1-3 or BMP-1-4 or the example of form include but not limited to, immunoblotting (such as, Western blotting, Dot blot), enzyme-linked immunosorbent assay (" ELISA "), radioimmunoassay (" RIA "), immunoprecipitation, affine method, immuno-chip etc.

According to the present invention, provide the method for diagnosing acute myocardial infarction (AMI) in individual human, it comprises the existence measuring BMP-1-4 in individual blood, wherein detects that BMP-1-4 indicates this individuality to have persistence AMI in blood.In preferred embodiments, BMP-1-4 antibody is for detecting the BMP-1-4 in individual blood.Such as, use suitable imaging system and the BMP-1-4 antibody being connected to suitable detectable label, the existence of the BMP-1-4 circulated in periphery can be detected in vivo.But, in the more preferred of the method for diagnosing the AMI in individual human, from individual human, obtaining blood sample and measure the existence of BMP-1-4 in vitro.

In typical immunoassay format, the blood sample obtained from individuality is contacted with BMP-1-4 antibody molecule.Using this area obtainable for detecting antibody-antigene in conjunction with any one in the multiple detection system of complex subsequently, detecting being formed in conjunction with complex between the BMP-1-4 protein that exists in BMP-1-4 antibody molecule and blood sample.

BMP-1-4 antibody for detecting or measure BMP-1-4 amount (that is, quantity) existed in blood can use in the solution, or alternately, can be fixed on the surface of any one of many kinds of solids substrate.BMP-1-4 antibody can be fixed thereon and include but not limited to for the solid substrate in method and composition described herein, magnetic substrate granule; Chromatography matrix or resin particle (such as, agarose); The surface in one or more holes of plastics assay plate (such as micropore assay plate); Solid substrate material flakes, the small pieces of such as plastics, nylon, timber, paper or other solid materials or bar, it can immerse blood sample or measures in solution or otherwise place with blood sample or measure solution and contact; And the surface of silicon (or other chip materials).BMP-1-4 antibody is fixed to the hole surface of microtitration plate or chip (such as, silicon, microscope slide etc.) surface, be allowed for the use of the form of the BMP-1-4 amount detecting or measure in one or more blood sample, be used in the semi-automation of conventional use in standard high throughput ELISA or biochip mensuration program or full-automatic device.Such device can be used in particular for the blood measuring a large amount of minimum volume with regard to the existence of BMP-1-4.

BMP-1-4 antibody can be fixed to the surface of solid substrate by any method, and described method retains the ability that this antibody is combined with BMP-1-4 when contacting with the blood sample containing BMP-1-4, to be formed in conjunction with complex.Such as, antibody or directly can be covalently attached to the surface of solids or linkers and be fixed to solid substrate by absorption (non-covalent adhesion) by antibody, and described linkers allows to use methods known in the art to tie antibody to solid substrate.

Detect the method in conjunction with complex comprising BMP-1-4 and BMP-1-4 antibody and preferably adopt the detection system using one or more signals to generate molecule (detectable label), described signal generates molecule and will generate easily through human eye detection, or easily pass through the signal that signal detection instrument (such as, spectrophotometer) detects or measures.Can be used for this type of signal detected in conjunction with complex to include but not limited to, such as, fluorescence signal as generated by the fluorescent dye or anthocyanidin molecule that directly or indirectly can be connected to BMP-1-4 antibody; Perceived color signal such as generated by the enzyme or coloured molecule (such as, pigment) that directly or indirectly can be connected to BMP-1-4 antibody; Radiated signal such as generated by the radiosiotope that directly or indirectly can be connected to BMP-1-4 antibody; And the optical signal such as generated by chemiluminescence or bio-luminescence system.The example of bio-luminescence system is luciferin-luciferase system, and wherein luciferase directly or indirectly can be connected to antibody, with under the existence of luciferin substrate, generates detectable optical signal.

Use the obtainable standard reagent in this area and scheme, detectable label directly or via linkers can be conjugated to BMP-1-4 antibody.Alternately, BMP-1-4 antibody can be unlabelled, and can't help the epi-position place of the first BMP-1-4 antibodies in conjunction with the second binding molecule (such as, antibody) of the BMP-1-4 in BMP-1-4 antibody or conjugated antigen-antibody binding complexes, may be used for generating detectable signal.This form is illustrated by standard sandwich immunoassay, wherein " capture antibody " (such as, BMP-1-4 antibody) binding purpose antigen is (such as, BMP-1-4), to be formed in conjunction with complex, and providing package is containing the second antibody (detection antibody) of detectable label subsequently, its in conjunction with capture antibody or can't help epi-position place that capture antibody combines with combine in conjunction with the object antigen in complex.Be to be understood that if second antibody is also BMP-1-4 antibody, then it must can't help epi-position that capture antibody is combined and combining in conjunction with epi-position complex exposing (can be close) of being formed between capture antibody and BMP-1-4 on BMP-1-4.Other changes of sandwich immunoassay are that skilled practitioner is known, and are applicable in method described herein.

In another kind mensuration form, the BMP-1-4 in blood sample uses BMP-1-4 antibody to adsorb with it or covalently bound test strip detects.This type of test strip provide detect or measure BMP-1-4 in blood sample facilitate method.Such as, manually or automatically by test strip to immerse in sample or by blood sample drops on the test strip, the test strip containing immobilization BMP-1-4 antibody can be made to contact with blood sample.Preferably, first test strip is immersed in sealer such as bovine serum albumin or other compositionss, to be reduced by the non-specific binding of potential interference molecule.When needing, this test strip can immerse any reagent further or contact with any reagent, described any reagent is that development or generate can detect or can needed for measuring-signal, describedly detects or measuring-signal instruction can comprise the existence in conjunction with complex with the BMP-1-4 of immobilization BMP-1-4 antibodies on this.This test strip visual observations subsequently, or by suitable detecting instrument reading, to determine existence or the amount of BMP 1-4 in sample.

The fraction that can adopt whole blood or whole blood for the method detected from the BMP-1-4 in the blood of individuality described herein, such as blood plasma or serum.In any particular assay form be use whole blood, blood plasma or serum or more even other Blood fractions final decision completely those of ordinary skill in the art understanding and judge in.Usually, blood plasma is preferred.

Use the standard method and the equipment that are used for obtaining blood sample from individuality, include but not limited to, the blood sample pipe of aseptic syringe needle, asepsis injector, sterile part evacuation is used for obtaining blood sample from individual human, is that bleeder and health care provider are well-known.

In order to Measurement accuracy (quantitatively) to derive from individual blood sample the BMP-1-4 level (amount, concentration) of (and, thus in the circulation of individuality), can use and measure graphic representation as described herein or use Practical computer teaching standard curve.Such as, mensuration described herein can be carried out one or more blood sample and a series of solution, described a series of solution contains the BMP-1-4 of concentration known, or the peptide of one or more epi-positions containing BMP-1-4 or peptide set (BMP-1-4 standard substance).The signal intensity obtained for each BMP-1-4 standard substance or magnitude are subsequently for building standard curve, and described standard curve makes the amount of signal intensity or magnitude and BMP-1-4 or concentration associate.The signal intensity from the sample of BMP-1-4 content the unknown or magnitude can be read out subsequently, with the corresponding BMP-1-4 level determining to exist in sample (amount, concentration) on standard curve.Preferably, BMP-1-4 in the sample of BMP-1-4 content the unknown determines horizontally through interpolation, that is, the standard curve region by generation or drafting between at least two BMP-1-4 standard points reads out the signal magnitude from the sample of BMP-1-4 content the unknown or intensity.More preferably but not optionally, can by the BMP-1-4 amount in extrapolation determination sample, wherein the magnitude of signal or intensity drop on the standard curve region that exceedes or exceed and to draw beyond two or more BMP-1-4 standard points or generate.

Method and composition described herein preferably adopts BMP-1-4 antibody as preferred BMP-1-4 binding partners, to detect the BMP-1-4 in the existence of the BMP-1-4 in blood sample or quantitative blood sample.But, it should also be understood that if this binding partners can be similar to for or be applicable to the method and compositions, then this type of method and composition can comprise the use of the BMP-1-4 binding partners except BMP-1-4 antibody molecule.

Can be assembled into test kit easily for the material detected needed for the BMP-1-4 in blood sample, described test kit allows health care provider to determine whether individuality has persistence acute myocardial infarction (AMI).In one embodiment, test kit of the present invention comprises BMP-1-4 antibody and description, and how described description instruction uses this test kit to carry out the mensuration of the BMP-1-4 detected in blood sample.In another embodiment, test kit can comprise the first antibody (capture antibody) in conjunction with BMP-1-4 antibody; Second antibody molecule (detection antibody), wherein said second antibody contains detectable label, and be combined with capture antibody or with can't help the BMP-1-4 epi-position that capture antibody is combined and combine; And how instruction uses this test kit to carry out detecting or the description of the mensuration of BMP-1-4 in quantitative blood sample.The BMP-1-4 antibody being used as capture antibody in test kit may be used in solution, maybe can be fixed in solid substrate, and surface of described solid substrate such as chip, pearl, test strip, micro titer plate well etc., it can contact with blood sample.Component capture antibody in test kit described herein and detection antibody can be packed in any one of multiple situation, and described situation is drying regime, non-hydrated state, lyophilised state, dewatering state or the hydration status in physiological buffered solution such as.For hydration, washing, blocking-up non-specific binding or come the detectable label on Autonomous test antibody signal generate solution also can be included in test kit described herein.Test kit can also comprise one or more devices obtaining blood sample from individual human body.This device includes but not limited to the blood sample pipe of aseptic nail, sterile needle, sterile needle and syringe and sterile-evacuate.

Other embodiment of the present invention and feature will be apparent according to following non-limiting example.

Embodiment

embodiment 1. uses LC/MS (" LC-MS "), by heparin sepharose affinity chromatography and identification of proteins, identifies BMP-1-3 and BMP-1-4 isotype from human plasma, instead of real skeletonization BMP.

The hemanalysis from people's object carried out about BMP-1-3 and BMP-1-4 isotype as discussed previously.See the people such as Grgurevic (2011); International publication number WO2008/011193.

Plasma collection

Blood sample from adult healthy volunteers and there is persistence acute myocardial infarction (AMI) patient collect.Blood sample is drawn in the syringe containing 3.8% sodium citrate, to form the anticoagulant of 1:9 and blood ratio (v/v).Blood plasma by centrifugal (15 minutes, with 3000 x g) obtain, and the aliquot of every part of adult's blood sample is for the preparation of the blood plasma stock solution merged.Before analysis by aliquot storage of samples at-80 DEG C.

affinity column purification

By human plasma (80 ml) 10 mM sodium phosphate buffers (pH 7) the dilution twice merged, and put on the 5 ml heparin sepharose posts (Amersham Pharmacia Biotech) previously balanced with 10 mM sodium phosphate buffers (pH 7).With 10 mM sodium phosphate buffers (pH 7) the elution of bound protein from post containing 1.0 M and 2.0 M NaCl.

ammonium sulfate precipitation

Saturated ammonium sulfate (" SAS ") is dropwise added Protein elution thing, turbine mixer is mixed to 35%(w/v) final concentration.Sample is kept 10 minutes on ice, and with 12,000 x gcentrifugal 5 minutes.Abandoning supernatant, and prepare agglomerate for the subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

the SDS-PAGE of protein purification and western blot analysis

According to the method for Laemmli ( nature, 227:680-685(1970)), use 10% gel to run agglomerate in standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE).After electrophoresis, a SDS-PAGE gel is transferred to celluloid, and other are directly with Coomassie brilliant blue (" CBB ") dyeing.First make nitrocellulose filter with for real skeletonization BMP such as BMP-7(Genera Research Laboratory) or the specific antibody of BMP-1 isotype together with incubation, and keep spending the night at 4 DEG C.The goat anti-mouse antibody that alkali phosphatase is puted together at room temperature is used as second antibody totally 1 hour.With 5 ml chromogenic substrates, film is developed the color.Other parts (0.1% CBB in 45% methanol, 10% acetic acid under standard dying operation of gel; At room temperature 30 minutes) dye with Coomassie brilliant blue (CBB).

Gel cutting is become to correspond to the section as each protein band by disclosing with CBB dyeing.With post-treatment gel slice, to determine which kind of protein is present in each section, use the method for the tryptic peptide discharged from each protein band by HPLC and mass spectrography (" MS ") analysis, use as by Olsen and Mann( proc. Natl. Acad. Sci. USA, 101:13417-13422(2004)) describe and by the people such as Grgurevic ( j. Nephrol., 20:311-319(2007)) the Nanoliter electrospray LC-MS interface revised.Hereafter indicate the aspect studying concrete relevant this method step to this.

gel trypsin digestion scheme

Band in gel cuts from the gel that CBB dyes, and uses trypsinization.In brief, gel chips is made to shrink 8 minutes with 100 μ l acetonitriles.Remove liquid, and make gel chips reflation 12 minutes with 100 μ l ammonium bicarbonate, and drying 10 minutes in SpeedVac subsequently.Add dithiothreitol, DTT (" DTT ", 100 μ l), and at 57 DEG C incubation 45 minutes.Gel chips 100 μ l acetonitriles are shunk 8 minutes at 57 DEG C, is rotated down and removes liquid.Iodoacetamide (100 μ l) is added each gel chips, and at room temperature in the dark without the need to stirring incubation 45 minutes.Add trypsin 10 μ l)/gel chips.Make gel chips centrifugal subsequently and reflation 10 minutes.Sample is incubated overnight in hot blender at 37 DEG C.

peptide extraction scheme

Sample is taken out from 37 DEG C of hot blenders.Add the solution (50 μ l) containing acetonitrile, water and formic acid.Make sample ultrasonic process 15 minutes.Supernatant is transferred to reserve tube, and adds 50 μ l acetonitriles.Make extract in SpeedVac, be dried to bone dry (about 40 minutes) under vacuo.With the 10 μ l solution containing water, methanol and formic acid, peptide is dissolved again.By sample ultrasonic process 5 minutes, and until when analyzing at being stored in-20 DEG C.

Mass spectrography

The following LC/MS (LC-MS) that passes through analyzes tryptic peptide.Use Nanoliter electrospray LC-MS interface (Proxeon Biosystems, Odense, Denmark), make Agilent 1100 nanoflow HPLC system (Agilent Technologies, Palo Alto, CA) with 7-Tesla LTQ-FT mass spectrograph (Thermo Electron, Bremen, Germany) coupling.Peptide is at self-control 75 μm of C ₁₈hPLC column is separated, and carries out mass spectral analysis with cation offline mode.Each measurement circulation is made up of following: mass spectrography (MS) scanning completely, is selected ionic monitoring (SIM) scanning subsequently, MS/MS and the MS/MS/MS scanning of three kinds of ions the strongest.This provide the usual peptide mass accuracy of 2 ppm, and from the other sequence information of MS/MS and MS/MS/MS fragment ion.Make the obtained spectrogram heart (centroided), and use Mascot search engine (Matrix Science) to search for for NCBInr data base.With trypsin specificity, carboxyamido methylate as fixing modification and oxidation methionine complete search as variable modification.The quality tolerance of 5 ppm and 0.6 Da is respectively used to MS and MS/MS spectrum.

result

For the blood from Healthy People individuality, LS-MS and immunoblotting assay disclose ten two (12) and plant tryptic peptide, and it is compared with NCBInr data base.Find that two kinds of peptides do not belong to any known real skeletonization BMP, but belong to the montage isotype 3(Swiss-Prot:P13497-2 of BMP-1-3 precursor; SEQ ID NO:2), i.e. precollagen C protein enzyme.The aminoacid sequence of two kinds of peptides is:

The aminoacid 653-660 of S-G-L-T-A-D-S-K(SEQ ID NO:2), Mascot score=36;

The aminoacid 280-289 of G-I-F-L-D-T-I-V-P-K(SEQ ID NO:2), Mascot score=26.

Other protein are not had to mate the peptide of identical group in NCBInr data base.Real skeletonization bmp protein matter is not detected at the molecular weight place of 100 kDa and 35 kDa by LS-MS or by immunoblotting.With previously found (WO 2008/011193 A2; The people such as Grgurevic (2011)) consistent, result shows that real skeletonization BMPs does not circulate usually in the blood of health adult, and BMP-1-3, i.e. precollagen C protein enzyme isoforms, be that the soluble proteome of normal human blood divides.

For the blood of people patient with persistence acute myocardial infarction, LS-MS and immunoblotting assay disclose the peptide of BMP-1-3 and two kinds of tryptic peptides compared with NCBInr data base.Find that two kinds of peptides belong to the aminoacid sequence of BMP-1-4 isotype (SEQ ID NO:3).The aminoacid sequence of two kinds of peptides is:

The aminoacid 203-216 of K-N-C-D-K-F-G-I-V-V-H-E-L-G(SEQ ID NO:3), Mascot score=51;

The aminoacid 244-256 of G-V-L-H-S-S-L-L-L-L-S-C-G(SEQ ID NO:3), Mascot score=64.

Therefore, although the blood of healthy individuals contains BMP-1-3(and do not contain other BMP-1 isotypes), the blood with the individual human of persistence acute myocardial infarction contains BMP-1-3 and BMP-1-4.This is that BMP-1-4 isotype protein is confirmed on protein level first, and display is present in the blood of the people patient of acute myocardial infarction, instead of in the blood of healthy individuals.Therefore, in the blood sample of individual human, the detection of BMP-1-4 indicates this individuality to have persistence acute myocardial infarction.

the location of embodiment 2. BMP-1-4 in human heart.

Use BMP-1-4 antibody, in the heart that BMP-1-4 has been positioned developmental Human embryo and Placenta Hominis (data do not show).In human adult heart section, BMP-1-4 protein detects in sarcostyle and myocardial cell, but does not detect (data do not show) in the tissue from other major organs multiple.The expression of result instruction BMP-1-4 isotype is unique relevant with function to the growth of human heart.

embodiment 3. is for studying the materials and methods of acute myocardial infarction.

antibody produces

Use the synthetic peptide fragment derived from BMP-1-3 and BMP-1-4 aminoacid sequence (being respectively SEQ ID NO:2 and SEQ ID NO:3), generate the polyclone for BMP-1-3 and BMP-1-4 and monoclonal antibody.

In order to produce the monoclonal antibody for BMP-1-3 protein, by the synthetic peptide of following aminoacid sequence in C-terminal region with BMP-1-3 protein, to mice immunisation:

The amino acid residue 972-986 of R-Y-T-S-T-K-F-Q-D-T-L-H-S-R-K(SEQ ID NO:2).

In order to produce monoclonal for BMP-1-4 protein and polyclonal antibody, by the synthetic peptide with following aminoacid sequence, to animal immunisation:

C-G-S-R-N-G-A-S-F-P-S-S-L-E-S-S-T-H-Q-A（SEQ ID NO:8）。

This peptide has the amino acid residue 255-274(SEQ ID NO:3 with BMP-1-4) identical aminoacid sequence, except the cysteine (Cys) at position 265 place has replaced with except serine (Ser).This formation stoping sulfydryl crosslinked, described sulfydryl is cross-linked the required immunogenicity site can blocked for generating anti-BMP-1-4 antibody.

Use the restructuring BMP-1-3(Genera Research Lab of enzyme-linked immunosorbent assay (ELISA) and purification) identify peptide-specific antibody.Make affinity matured antibody.

Above-mentioned peptide immunity inoculation Balb/C mice is used, from ProMab(Richmond, California, U.S. in the hybridoma operation of manufacturer) obtain monoclonal antibody for BMP-1-3 and BMP-1-4.The cutting of use standard measures, and the suppression that the precollagen mediated by BMP-1-3 or dentin extracellular matrix (DMP-1) are cut, confirms the Neutralization effect (data do not show) of BMP-1-3 antibody.See such as, the people (1996) such as the people such as Kessler (1996) and Li (precollagen 1 cutting measures); The people (2004) (DMP-1 cuts mensuration) such as the people such as Qin (2003) and Steiglitz.Shown in research as described below, BMP-1-3 mAb and BMP-1-4 mAb all effectively treats acute myocardial infarction in the rat model of disease.

the rat model of acute myocardial infarction

Research described herein adopts experimental acute myocardial infarction model in rats.This model presents those the pathophysiology change being similar to and occurring after acute myocardial infarction in people, and is select to change for studying the morphology and function making can occur after infarction the model dropping to minimum Results.See such as, the people such as Zornoff (2009).Originally six months large Sprague-Dawley rats are raised in steady temperature (25 DEG C) with day and night under photoperiodic standard conditions.The combination of the xylazine (0.6 ml/kg, Rompun, Bayer AG, Leverkusen, Germany) that the male rat intraperitoneal of weighing 250-300 gram is used and ketamine (Narketan, 0.8 ml/kg, Chassot GmbH, Germany) is anaesthetized.After the 4th intercostal space place carries out left thoracotomy, cut pericardium.Heart exteriorization by the transverse direction pressing of chest.Ligature (6/0 Ethilon suture, Ethicon, Somerville, New Jersey, the U.S.) is placed in around left coronary artery main stem, close to its starting point and between left atrium and pulmonary artery ditch.Subsequently, send heart back to thoracic cavity fast, and with positive airway pressure, lung is expanded.

the measurement of Plasma CK-MB and troponin t

By measuring the blood plasma level of two kinds of cardiac markers and MB creatine kinase (" CK-MB ") and troponin (" Tn-T "), in the rat of acute myocardial infarction (AMI) of implementing ligation induction as described herein, assess myocardial cell injury and necrosis, described two kinds of cardiac marker are the damage to cardiac tissue labellings determined.The level rising instruction damage to cardiac tissue of CK-MB and Tn-T, comprises the damage to cardiac tissue from acute myocardial infarction in blood.Blood sample extracts from the Ocular Vessels clump of animal, and is collected in the pipe of heparinization.Sample is rapidly with 2000 x gcentrifugal 15 minutes, until when taking to measure.Enzyme-linked immunosorbent assay (ELISA) is used to measure two kinds of AMI labellings.

Use commercial ELISA Kit (Troponin T hs STAT, Roche Diagnostics, Mannheim, Germany), in following research, determine the troponin t(" Tn-T " in blood sample) level.This specific ELISA is quantitative sandwich enzyme immunoassay (EIA), and it adopts has by the microtitration plate for the pre-coated hole of the specific antibody of Tn-T.Standard and sample are added in hand-hole, and any Tn-T existed in standard or sample is combined by sessile antibody.After any unconjugated material of removal, the same antibody for the specific biotin-conjugated of Tn-T is added in hand-hole.After washing, the horseradish peroxidase (HRP) puted together by avidin adds in hand-hole.Subsequently by HRP zymolyte TMB(3,3', 5,5' tetramethyl benzidine) add in hand-hole, react with initial HRP.In incubation period process, HRP reaction generates measures proportional color with the Tn-T be combined with micro titer plate well in initial step.By stopping solution (sulfuric acid solution) cessation reaction, and the color of reactant mixture in the 450 each holes of spectrophotometer measurement, nm place, nm ± 2.The Tn-T concentration in sample is determined subsequently by the O.D. of comparative sample and standard curve.

CK-MB level in blood sample also uses ELISA kit (Creatine Kinase-MB, CKMBL, Roche Diagnostics, Indianapolis, Indiana, the U.S.) to determine.

echocardiography

All animals (rat) in following research all experience echocardiography under anaesthesia.Animal light anaesthesia is made with ketamine and xylazine combination.Performing two dimension (2D) guides lower M-mode transthoracic echocardiography to check.For M-mode record, parasternal short axis view is for making the 2D cardiac imaging on mitral level.Left ventricle (LV) volume is calculated via 2D measurement by formula.Measure following M-mode to measure: LV(left ventricle) wall thickness (being respectively LVPWd and LVPWs) and relaxing period and systole interventricular septum size (being respectively IVSd and IVSs) after relaxing period and systole internal diameter (being respectively LVIDd and LVIDs), LV relaxing period and systole.Measure according to these, derivative ejection fraction (EF) and Fractional shortening (ES).Ultrasoundcardiogram performs three times by two different doctors to every animal, and result is rendered as meansigma methods.

pET data acquisition and data analysis

Positron emission tomography (PET) is medicine imaging technique, and it, by following the tracks of the distribution of radiolabeled bioactive molecule in room and time be expelled in laboratory animal, produces three-dimensional (3D) image of the specific function process in body.The gamma-rays pair of this systems axiol-ogy by producing from the positron annihilation of the positron-emitting radioactive nucleic (tracer) being connected to bioactive molecule, and the 3-D view of tracer concentration is in vivo reconstructed subsequently by computer analysis.In acute myocardial infarction described herein research, bioactive molecule is fluorodeoxyglucose (FDG, fluorodeoxyglucose), its be can intravenous injection to the glucalogue in laboratory animal.FDG is absorbed by function cardiac muscular tissue instead of non-functional ischemic myocardial tissue.This technology relies on (will be definite contrary in their barycenter frame) coincidence that the photon of movement is right on approximately contrary direction and detects.

The process occurred through relatively long-time section is paid close attention in research described herein.Especially, for every animal in acute myocardial infarction research, obtain the image measured to obtain when three time points: (1) on pretreatment, to determine the baseline of every animal, (2) after the ligation producing ischemia effect (1 week), and after surgery more for a long time (1 month).By this way, contain all stages of recovery process: baseline (normal ingestion), acute stage (after surgery immediately) and long-term recovery.

Use 3.3 versions of PMOD software to perform data analysis, it is synchronous and correction about the input from ClearPet photographing unit.

Analysis is divided into two steps: (1) qualitative analysis and (2) quantitative analysis.In order to determine quantitative effect, designing following operation: first, determining the time average that can accept time frame.Subsequently, FUSION PMOD program is used for recording different measuring (making it reach same position) altogether.For acute myocardial infarction experiment, all measurements are all total to record with base line measurement.Next, obtain the 3D rendering about the heart of each experiment with the 3D PMOD program of SURFACE pattern formula, but surperficial threshold value is become higher value from minima, to describe the isoactivity line in all animals.

the embodiment 4. CK-MB enzyme analysis had in the rat plasma of acute myocardial infarction of the monoclonal antibody process for BMP-1-3.

This determines the blood plasma level of MB creatine kinase (CK-MB) protein in the rat of the acute myocardial infarction with ligation induction, and described rat processes by the monoclonal antibody (BMP-1-3 mAb) for BMP-1-3 with rear before surgery.Altogether use 16 rats.Treatment group animal being divided into the matched group be made up of 6 animals and being made up of 10 rats, described 10 rats BMP-1-3 monoclonal antibody (15 μ g/kg) carries out pretreatment.After surgery, surviving animals is divided into two groups: (1) has the control rats coronarius (n=4) of ligation, and (2) there is the coronary artery of ligation and the every day between the period 1 with the rat (n=7) of BMP-1-3 monoclonal antibody process.Different time points collect blood: before surgery, and after surgery first day, second day, the 3rd day and the 7th day.As shown in Figure 2, CK-MB value before ligation is similar, and 24 hours after surgery (1 day), this value lower in the rat of antibody treatment (with 376.7 U/L in the group of BMP-1-3 mAb process relative to 459 U/L in undressed matched group).Second day time, CK-MB is 621.8 in undressed control rats, and it is only 441.9(p<0.05 in the rat with BMP-1-3 mAb process).Afterwards during time point, compared with undressed control rats, CK-MB value is also lower in the rat with BMP-1-3 mAb process.See Fig. 2.

Result instruction BMP-1-3 is the therapeutic target of acute myocardial infarction, and the antibody used for BMP-1-3 is for the effective therapy with regard to acute myocardial infarction individuality.

the embodiment 5. CK-MB enzyme analysis had in the rat plasma of acute myocardial infarction of the polyclonal antibody process for BMP-1-4.

Altogether use 20 rats in this experiment.The CK-MB level when different time points in rat blood of measurement: coronary artery ligation operation consent and after ligation first day, second day, the 3rd day, the 6th day and the 7th day).Ten rat BMP-1-4 polyclonal antibodies (15 μ g/kg) carry out pretreatment.After surgery, the rat of coronary artery ligation of surviving is divided into two groups: (1) without the control rats (n=8) of acute myocardial infarction with ligation induction for the treatment of, and (2) have ligation induction acute myocardial infarction and with the rat (n=6) of BMP-1-4 polyclonal antibody process.As shown in Figure 3, compared with undressed control rats, 2 days after surgery, significantly reduce the CK-MB blood serum values (in such as matched group 563.8 relative to 441.5 in the rat of antibody treatment) in rat with the process of BMP-1-4 polyclonal antibody.Afterwards during time point, compared with undressed control rats, CK-MB value in the rat with the process of BMP-1-4 polyclonal antibody also lower (such as the 7th day time, 395.5 in matched group compares with 237.2 in the rat with the process of BMP-1-4 polyclonal antibody).

Result instruction BMP-1-4 is the therapeutic target of acute myocardial infarction, and the antibody used for BMP-1-4 is the effective therapy being used for the treatment of acute myocardial infarction.

the embodiment 6. troponin t had in the rat plasma of acute myocardial infarction of the combined treatment of BMP-1-3 and BMP-1-4 monoclonal antibody analyzes.

The troponin t(" Tn-T " had in the rat of the acute myocardial infarction (AMI) of induction is followed the tracks of in this research) level, in the ligation operation front and rear of induction AMI, described rat is combined into row relax with BMP-1-3 mAb's and BMP-1-4 mAb.21 rats are altogether used in this research.Before coronary artery ligation, seven rat combination (BMP-1-3 antibody+BMP-1-4 antibody; Often kind of antibody 15 μ g/kg) carry out pretreatment, and 14 rats keep untreated (matched group).After 24 hours, the surgical ligation of rat experience left coronary artery.Surviving animals is divided into two groups: (1) has the control rats coronarius (n=6) of ligation, and the rat (n=3) with the AMI through induction of (2) BMP-1-3 mAb+BMP-1-4 mAb process.24 and 48 hours after surgery, the animal of antibody treatment accepted often kind of antibody of 15 μ g/kg.Blood is collected when different time points: before surgery, first day, second day, the 3rd day and the 6th day (Fig. 4) after ligation operation.Relative to the undressed control animal with AMI, the combination display of antibody reduces the remarkable efficacy of serum T n-T level.At first day, during second day and the 3rd day, this value in undressed control rats is 7.8,3.26,1.18, and in the animal of antibody treatment, this value is 5.72,1.63 and 0.24.See Fig. 4.

The combination treatment of result instruction BMP-1-3 mAb and BMP-1-4 mAb effectively treats acute myocardial infarction.

embodiment 7. has the echocardiography of the cardiac function in the rat of acute myocardial infarction.

By the control rats of undressed coronary artery ligation and the rat of coronary artery ligation that processes by BMP-1-3 monoclonal antibody (15 μ g/kg) with the open in-heart operation under pulsating of M-mode, the functional outcome of further Effect of Anti autogenic therapy and fibrosis are formed.19 rats are used for the research of this long term follow-up altogether.After surgery, survival rats is divided into three groups: (1) sham operated rats: normal, healthy animal (n=3), (2) matched group: the undressed rat (n=4) with the acute myocardial infarction through induction, (3) treatment group: between the period 1 before surgery and after surgery, with the rat (n=7) with the acute myocardial infarction through induction of BMP-1-3 monoclonal antibody process.Within 45 days, perform ultrasoundcardiogram after surgery.Use the analysis of healthy rat, according to limiting meansigma methods as follows: IVSd=1.1 mm, LVIDd=5.3 mm, LVPWd=1.77 mm, IVSs=2.3 mm, LVIDs=2.7 mm, LVPWs=2.4 mm, EF=85.5%, FS=49.1%.The control rats of acute myocardial infarction had through induction has the remarkable reduction in function, and this occurs in echocardiographic parameters:: IVSd=1 mm, LVPWd=1.73 mm, IVSs=1.1 mm, EF=68.8%, FS=33.9%.With BMP-1-3 monoclonal antibody process (treatment) cardiac function enhancing: IVSd=1.38 mm, LVIDd=5.9 mm, IVSs=2.7 mm, EF=88.2%, FS=52.9%.See table 1.

Table 1. is in the animal model of acute myocardial infarction, and the cardiac dimensions of rat and the ultrasoundcardiogram of function are measured.

False=sham-operation rat; The undressed rat of the acute myocardial infarction of contrast=enforcement ligation induction; The BMP-1-3 monoclonal antibody process of the rat of the acute myocardial infarction of BMP1-3 mAb=enforcement ligation induction; * the statistical significance of p<0.05(compared with control rats).

(PET) data acquisition of embodiment 8. positron emission tomography and data analysis.

In this experiment, in the rat model of acute myocardial infarction, 20 rats are scanned by PET before surgery.Before surgery, rat is divided into control rats (n=10) and the animal (n=10) with BMP-1-3 mAb process.After surgery, mortality rate is 50% in control rats, and before surgery with being 30% in the rat of BMP-1-3 mAb process.Subsequently the 2nd, 7 and 14 day time, with BMP-1-3 mAb with the dosage process rat of 15 μ g/kg.Except before surgery first time PET scan except, rat also first week with the laggard line scanning of first month, with the impact of the progress and treatment of assessing infarction.Representative cardiac image from the animal of matched group and BMP-1-3 mAb processed group is shown in Fig. 5.After first week, two groups are all presented at FDG picked-up (0.36 relative to 0.38) reduced in infarcted region.With in the rat of BMP-1-3 mAb process after one month, FDG picked-up in infarcted region recovers (0.42), indicate substantially reinventing and regenerating of the function cardiac muscular tissue in former infarcted region, and in undressed control rats, picked-up still very low (0.36), indicates non-functional scar tissue.See Fig. 5.

the histologic analysis of embodiment 9. cardiac muscle after acute myocardial infarction.

Histologic analysis is performed, to evaluate the effect processed by BMP-1-3 monoclonal antibody (BMP-1-3 mAb) to the cardiac muscle of the rat with the acute myocardial infarction (AMI) that ligation is induced.Rat (n=16) is divided into the matched group be made up of six animals and the treatment group be made up of 10 animals, described 10 animals BMP-1-3 mAb(15 μ g/kg) carry out pretreatment.After surgery, surviving animals (n=11) is divided into two groups: (1) has control rats (n=4 coronarius of ligation, do not use the pretreatment of BMP-1-3 mAb), and (2) every day with BMP-1-3 pretreatment and subsequently between the period 1 is with the rat coronarius (n=7) with ligation of BMP-1-3 mAb process.

Take out the cardiac muscular tissue (about 2 mm of thickness) of the left ventricle from AMI rat.Sample is fixed 72 hours at 4% pre-cooling paraformaldehyde, and embedding is used for Histological research in paraffin.Paraffin-embedded tissue slice is become about 5 μm of slabs.Section standard hematoxylin and Yihong (" H & E ") dye, to disclose cellular component, and with Picro-Sirius red (and picric acid) dyeing, to identify that viscose basic stitch is accumulated.Image manifests under an optical microscope.

Fig. 6 shows for having the undressed control rats of AMI and the rat with AMI with BMP-1-3 mAb process, in coronary artery ligation afterwards from the microphotograph of the histologic analysis of the cardiac muscle of rat.When Fig. 6 A shows one week after the left coronary artery ligation of inducing AMI, from the heart sections (in Fig. 6 A 4X amplification) of the infarcted region of undressed control rats heart.Fig. 6 B shows sirius red dyeing (under 20X amplification) of the tissue of the rectangular area in Fig. 6 A, indicates early stage collagen deposition.See the arrow in Fig. 6 B.Fig. 6 C shows with h and E dyeing, from the Cardiac muscle sections of undressed rat control with AMI, disclose after one month by the cardiac muscle fiber damaged around residual fibers scar tissue.See the arrow in Fig. 6 C.Fig. 6 D shows the heart sections in the cardiac infarction district from rat, described rat before the left coronary artery ligation of induction AMI with BMP-1-3 mAb(15 μ g/kg) process, and subsequently between period 1 after surgery every day with BMP-1-3 mAb process.Fibrosis region after AMI is significantly less than observe in control rats that.See the arrow in Fig. 6 D.Fig. 6 E shows the more magnification at high multiple rate in the region by the arrow instruction in Fig. 6 D, discloses new regenerated muscle fibers speckle.See the arrow in Fig. 6 E.Fig. 6 F shows the more magnification at high multiple in the region by the instruction of arrow in Fig. 6 E, discloses the cell around the muscle fiber and fibrous tissue that are recently formed, its with observe in the tissue from undressed control rats that compared with obviously more not fine and close.

The histologic analysis instruction of the heart muscle tissue after AMI significantly reduces the size of cicatrix with BMP-1-3 mAb process, and promotes that in original infarcted region, have the myofibrillar tuberosity recently formed is formed.

In a word, the result of above-described embodiment clearly indicates uses the progress that the antibody for BMP-1-3 and/or the antibody for BMP-1-4 effectively reduce the original infarcted region in individual heart, described individuality has persistence acute myocardial infarction, and effectively promoting the tissue remodeling in original infarcted region, having than that significantly higher quality when there is not antibody treatment and the reparation and the scar tissue that more have function to be formed.

The all patents quoted in text above, application and open to be incorporated to all by reference herein.

Other changes of the present invention described herein and embodiment will be apparent at present for those skilled in the art, and not deviate from disclosure of the present invention or following claims.

Sequence table

<110> GENERA ISTRAZIVANJA d.o.o.

VUKICEVIC, Slobodan

GRGUREVIC, Lovorka

DUMIC-CULE, Ivo

<120> is used for the treatment of the method and composition with diagnosing acute myocardial infarction

<130> ZGR-416.1PCT

<140> PCT/US2013/038294

<141> 2013-04-25

<150> 61/638,373

<151> 2012-04-25

<150> 61/638,424

<151> 2012-04-25

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 730

<212> PRT

<213> homo sapiens

<400> 1

Met Pro Gly Val Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu Leu Leu

1 5 10 15

Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr Tyr Asp Leu

20 25 30

Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys Asp Pro Cys Lys

35 40 45

Ala Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp Glu Glu Asp Leu Arg

50 55 60

Ala Phe Gln Val Gln Gln Ala Val Asp Leu Arg Arg His Thr Ala Arg

65 70 75 80

Lys Ser Ser Ile Lys Ala Ala Val Pro Gly Asn Thr Ser Thr Pro Ser

85 90 95

Cys Gln Ser Thr Asn Gly Gln Pro Gln Arg Gly Ala Cys Gly Arg Trp

100 105 110

Arg Gly Arg Ser Arg Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg

115 120 125

Val Trp Pro Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr

130 135 140

Gly Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His Trp Glu Lys

145 150 155 160

His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile

165 170 175

Val Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg

180 185 190

Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe

195 200 205

Gly Ile Val Val His Glu Leu Gly His Val Val Gly Phe Trp His Glu

210 215 220

His Thr Arg Pro Asp Arg Asp Arg His Val Ser Ile Val Arg Glu Asn

225 230 235 240

Ile Gln Pro Gly Gln Glu Tyr Asn Phe Leu Lys Met Glu Pro Gln Glu

245 250 255

Val Glu Ser Leu Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met His Tyr

260 265 270

Ala Arg Asn Thr Phe Ser Arg Gly Ile Phe Leu Asp Thr Ile Val Pro

275 280 285

Lys Tyr Glu Val Asn Gly Val Lys Pro Pro Ile Gly Gln Arg Thr Arg

290 295 300

Leu Ser Lys Gly Asp Ile Ala Gln Ala Arg Lys Leu Tyr Lys Cys Pro

305 310 315 320

Ala Cys Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro

325 330 335

Glu Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile

340 345 350

Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp

355 360 365

Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp

370 375 380

Gly Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly Ser Lys

385 390 395 400

Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe

405 410 415

Arg Ser Ser Ser Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu

420 425 430

Ala Ile Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser

435 440 445

Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg

450 455 460

Ile Gln Val Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser Phe

465 470 475 480

Glu Ile Glu Arg His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg

485 490 495

Asp Gly His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr

500 505 510

Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys

515 520 525

Phe Val Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe

530 535 540

Phe Lys Glu Val Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu

545 550 555 560

Gln Arg Cys Leu Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro

565 570 575

Gly Tyr Glu Leu Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala Cys Gly

580 585 590

Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro

595 600 605

Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val Ala Pro

610 615 620

Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr Glu Gly

625 630 635 640

Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val Arg Ser Gly Leu Thr

645 650 655

Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser Glu Lys Pro Glu

660 665 670

Val Ile Thr Ser Gln Tyr Asn Asn Met Arg Val Glu Phe Lys Ser Asp

675 680 685

Asn Thr Val Ser Lys Lys Gly Phe Lys Ala His Phe Phe Ser Glu Lys

690 695 700

Arg Pro Ala Leu Gln Pro Pro Arg Gly Arg Pro His Gln Leu Lys Phe

705 710 715 720

Arg Val Gln Lys Arg Asn Arg Thr Pro Gln

725 730

<210> 2

<211> 986

<212> PRT

<213> homo sapiens

<400> 2

Met Pro Gly Val Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu Leu Leu

1 5 10 15

Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr Tyr Asp Leu

20 25 30

Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys Asp Pro Cys Lys

35 40 45

Ala Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp Glu Glu Asp Leu Arg

50 55 60

Ala Phe Gln Val Gln Gln Ala Val Asp Leu Arg Arg His Thr Ala Arg

65 70 75 80

Lys Ser Ser Ile Lys Ala Ala Val Pro Gly Asn Thr Ser Thr Pro Ser

85 90 95

Cys Gln Ser Thr Asn Gly Gln Pro Gln Arg Gly Ala Cys Gly Arg Trp

100 105 110

Arg Gly Arg Ser Arg Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg

115 120 125

Val Trp Pro Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr

130 135 140

Gly Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His Trp Glu Lys

145 150 155 160

His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile

165 170 175

Val Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg

180 185 190

Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe

195 200 205

Gly Ile Val Val His Glu Leu Gly His Val Val Gly Phe Trp His Glu

210 215 220

His Thr Arg Pro Asp Arg Asp Arg His Val Ser Ile Val Arg Glu Asn

225 230 235 240

Ile Gln Pro Gly Gln Glu Tyr Asn Phe Leu Lys Met Glu Pro Gln Glu

245 250 255

Val Glu Ser Leu Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met His Tyr

260 265 270

Ala Arg Asn Thr Phe Ser Arg Gly Ile Phe Leu Asp Thr Ile Val Pro

275 280 285

Lys Tyr Glu Val Asn Gly Val Lys Pro Pro Ile Gly Gln Arg Thr Arg

290 295 300

Leu Ser Lys Gly Asp Ile Ala Gln Ala Arg Lys Leu Tyr Lys Cys Pro

305 310 315 320

Ala Cys Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro

325 330 335

Glu Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile

340 345 350

Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp

355 360 365

Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp

370 375 380

Gly Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly Ser Lys

385 390 395 400

Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe

405 410 415

Arg Ser Ser Ser Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu

420 425 430

Ala Ile Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser

435 440 445

Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg

450 455 460

Ile Gln Val Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser Phe

465 470 475 480

Glu Ile Glu Arg His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg

485 490 495

Asp Gly His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr

500 505 510

Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys

515 520 525

Phe Val Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe

530 535 540

Phe Lys Glu Val Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu

545 550 555 560

Gln Arg Cys Leu Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro

565 570 575

Gly Tyr Glu Leu Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala Cys Gly

580 585 590

Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro

595 600 605

Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val Ala Pro

610 615 620

Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr Glu Gly

625 630 635 640

Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val Arg Ser Gly Leu Thr

645 650 655

Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser Glu Lys Pro Glu

660 665 670

Val Ile Thr Ser Gln Tyr Asn Asn Met Arg Val Glu Phe Lys Ser Asp

675 680 685

Asn Thr Val Ser Lys Lys Gly Phe Lys Ala His Phe Phe Ser Asp Lys

690 695 700

Asp Glu Cys Ser Lys Asp Asn Gly Gly Cys Gln Gln Asp Cys Val Asn

705 710 715 720

Thr Phe Gly Ser Tyr Glu Cys Gln Cys Arg Ser Gly Phe Val Leu His

725 730 735

Asp Asn Lys His Asp Cys Lys Glu Ala Gly Cys Asp His Lys Val Thr

740 745 750

Ser Thr Ser Gly Thr Ile Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro

755 760 765

Ser Lys Lys Glu Cys Thr Trp Ala Ile Ser Ser Thr Pro Gly His Arg

770 775 780

Val Lys Leu Thr Phe Met Glu Met Asp Ile Glu Ser Gln Pro Glu Cys

785 790 795 800

Ala Tyr Asp His Leu Glu Val Phe Asp Gly Arg Asp Ala Lys Ala Pro

805 810 815

Val Leu Gly Arg Phe Cys Gly Ser Lys Lys Pro Glu Pro Val Leu Ala

820 825 830

Thr Gly Ser Arg Met Phe Leu Arg Phe Tyr Ser Asp Asn Ser Val Gln

835 840 845

Arg Lys Gly Phe Gln Ala Ser His Ala Thr Glu Cys Gly Gly Gln Val

850 855 860

Arg Ala Asp Val Lys Thr Lys Asp Leu Tyr Ser His Ala Gln Phe Gly

865 870 875 880

Asp Asn Asn Tyr Pro Gly Gly Val Asp Cys Glu Trp Val Ile Val Ala

885 890 895

Glu Glu Gly Tyr Gly Val Glu Leu Val Phe Gln Thr Phe Glu Val Glu

900 905 910

Glu Glu Thr Asp Cys Gly Tyr Asp Tyr Met Glu Leu Phe Asp Gly Tyr

915 920 925

Asp Ser Thr Ala Pro Arg Leu Gly Arg Tyr Cys Gly Ser Gly Pro Pro

930 935 940

Glu Glu Val Tyr Ser Ala Gly Asp Ser Val Leu Val Lys Phe His Ser

945 950 955 960

Asp Asp Thr Ile Thr Lys Lys Gly Phe His Leu Arg Tyr Thr Ser Thr

965 970 975

Lys Phe Gln Asp Thr Leu His Ser Arg Lys

980 985

<210> 3

<211> 302

<212> PRT

<213> homo sapiens

<400> 3

Met Pro Gly Val Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu Leu Leu

1 5 10 15

Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr Tyr Asp Leu

20 25 30

Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys Asp Pro Cys Lys

35 40 45

Ala Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp Glu Glu Asp Leu Arg

50 55 60

Ala Phe Gln Val Gln Gln Ala Val Asp Leu Arg Arg His Thr Ala Arg

65 70 75 80

Lys Ser Ser Ile Lys Ala Ala Val Pro Gly Asn Thr Ser Thr Pro Ser

85 90 95

Cys Gln Ser Thr Asn Gly Gln Pro Gln Arg Gly Ala Cys Gly Arg Trp

100 105 110

Arg Gly Arg Ser Arg Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg

115 120 125

Val Trp Pro Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr

130 135 140

Gly Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His Trp Glu Lys

145 150 155 160

His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile

165 170 175

Val Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg

180 185 190

Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe

195 200 205

Gly Ile Val Val His Glu Leu Gly His Val Val Gly Phe Trp His Glu

210 215 220

His Thr Arg Pro Asp Arg Asp Arg His Val Ser Ile Val Arg Glu Asn

225 230 235 240

Ile Gln Pro Gly Val Leu His Ser Ser Leu Leu Leu Leu Ser Cys Gly

245 250 255

Ser Arg Asn Gly Ala Ser Phe Pro Cys Ser Leu Glu Ser Ser Thr His

260 265 270

Gln Ala Leu Cys Trp Thr Gly Leu Phe Leu Arg Pro Ser Pro Phe Pro

275 280 285

Arg Leu Pro Leu Ala Ala Pro Arg Thr Leu Arg Ala Gly Val

290 295 300

<210> 4

<211> 4

<212> PRT

<213> homo sapiens

<220>

The still unclassified characteristic of <221>

<222> (3)..(3)

<223> X is arbitrary amino acid

<400> 4

Phe Gly Xaa Gly

1

<210> 5

<211> 9

<212> PRT

<213> homo sapiens

<220>

The still unclassified characteristic of <221>

<222> (2)..(9)

<223> X is arbitrary amino acid

<400> 5

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5

<210> 6

<211> 5

<212> PRT

<213> homo sapiens

<400> 6

Leu Glu Trp Ile Gly

1 5

<210> 7

<211> 4

<212> PRT

<213> homo sapiens

<220>

The still unclassified characteristic of <221>

<222> (3)..(3)

<223> X is arbitrary amino acid

<400> 7

Trp Gly Xaa Gly

1

<210> 8

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> is from the modified fragments of peptides of BMP-1-4

<400> 8

Cys Gly Ser Arg Asn Gly Ala Ser Phe Pro Ser Ser Leu Glu Ser Ser

1 5 10 15

Thr His Gln Ala

20

Claims

1. treat the method for the acute myocardial infarction in individuality, it comprises the combination of using the antibody for BMP-1-3, the antibody for BMP-1-4 or the antibody for BMP-1-3 and the antibody for BMP-1-4 to this individuality.

2. method according to claim 1, it comprises uses antibody for BMP-1-3 to this individuality.

3. method according to claim 1, it comprises uses antibody for BMP-1-4 to this individuality.

4. for BMP-1-3 antibody, for the antibody of BMP-1-1 or the combination product of two kinds of antibody, it is used for the treatment of the acute myocardial infarction in individuality.

5. antibody according to claim 4, it is made up of the antibody for BMP-1-3.

6. antibody according to claim 4, it is made up of the antibody for BMP-1-4.

7. antibody according to claim 4, it is made up of the combination of the antibody for BMP-1-3 and the antibody for BMP-1-4.

8. determine the individual method whether with persistence acute myocardial infarction, it comprises the existence that mensuration had previously derived from BMP-1-4 in the blood sample of this individuality, and wherein in this blood sample, the existence of BMP-1-4 indicates this individuality to have persistence acute myocardial infarction.

9. method according to claim 8, the step wherein measuring the existence of BMP-1-4 in described blood sample comprises makes described blood sample contact with BMP-1-4 binding partners, be formed between the BMP-1-4 that exists in described binding partners and described blood sample in conjunction with complex.

10. method according to claim 9, what formed between the BMP-1-4 wherein existed in described binding partners and described blood sample is detected by the detectable label relevant to described BMP-1-4 binding partners in conjunction with complex.

11. methods according to claim 9, what formed between the BMP-1-4 wherein existed in described BMP-1-4 binding partners and described blood sample is detected by following in conjunction with complex: add with described BMP-1-4 binding partner binds or with described in conjunction with complex in the antibody that combines of the described BMP-1-4 that exists, and detect described antibody by the detectable label existed on described antibody.

12. the method according to Claim 8 any one of-11, wherein said BMP-1-4 binding partners is the antibody in conjunction with BMP-1-4.

13. for the antibody of BMP-1-4 as the purposes of the biomarker for diagnosing acute myocardial infarction.

14. the amino acid residue 972-986 for peptide based immunogens R-Y-T-S-T-K-F-Q-D-T-L-H-S-R-K(SEQ ID NO:2) the anti-BMP-1-3 antibody that generates.

15. for peptide based immunogens C-G-S-R-N-G-A-S-F-P-S-S-L-E-S-S-T-H-Q-A(SEQ ID NO:8) the anti-BMP-1-4 antibody that generates.

16. pass through as being preserved in DSMZ, registration number hybridoma produce anti-BMP-1-3 antibody.